U.S. patent application number 11/796259 was filed with the patent office on 2009-04-23 for methods of treating inflammation.
Invention is credited to Stefan Brocke, Amanda Dall, Hongli Dong, Paul Epstein.
Application Number | 20090105281 11/796259 |
Document ID | / |
Family ID | 40564075 |
Filed Date | 2009-04-23 |
United States Patent
Application |
20090105281 |
Kind Code |
A1 |
Epstein; Paul ; et
al. |
April 23, 2009 |
Methods of treating inflammation
Abstract
The invention features compositions and methods for treating
inflammation and other immune-related disorders.
Inventors: |
Epstein; Paul; (Weatogue,
CT) ; Brocke; Stefan; (West Hartford, CT) ;
Dong; Hongli; (Glastonbury, CT) ; Dall; Amanda;
(Farmington, CT) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY AND POPEO, P.C
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
40564075 |
Appl. No.: |
11/796259 |
Filed: |
April 27, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60795652 |
Apr 27, 2006 |
|
|
|
Current U.S.
Class: |
514/262.1 ;
435/18; 435/29; 435/6.16 |
Current CPC
Class: |
A61P 3/10 20180101; A61P
25/00 20180101; C12Q 1/6886 20130101; C12Q 2600/158 20130101; A61K
31/519 20130101; A61P 29/00 20180101; A61P 35/00 20180101; G01N
33/5029 20130101; G01N 2333/916 20130101 |
Class at
Publication: |
514/262.1 ;
435/29; 435/18; 435/6 |
International
Class: |
A61K 31/519 20060101
A61K031/519; A61P 29/00 20060101 A61P029/00; A61P 3/10 20060101
A61P003/10; A61P 25/00 20060101 A61P025/00; A61P 35/00 20060101
A61P035/00; C12Q 1/02 20060101 C12Q001/02; C12Q 1/34 20060101
C12Q001/34; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. A method of inhibiting migration of an activated lymphocyte to a
site of inflammation, comprising contacting said lymphocyte with a
composition that preferentially inhibits cyclic nucleotide
phosphodiesterase (PDE) 8.
2. The method of claim 1, wherein said lymphocyte comprises a T
helper type 1 phenotype or a T helper type 17 phenotype.
3. The method of claim 1, wherein said composition comprises
dipyridamole or a derivative thereof.
4. The method of claim 1, wherein said composition further
comprises an inhibitor of PDE4, an inhibitor of PDE7 or both an
inhibitor of PDE4 and an inhibitor of PDE7.
5. An anti-inflammatory composition comprising a synergistic
combination of a PDE8 inhibitor and a PDE4 inhibitor.
6. A method of reducing or preventing a symptom associated with a
disease of the immune system comprising administering to a subject
in need thereof a composition comprising an inhibitor of PDE8 in an
amount effective to reduce lymphocyte chemotaxis in said
subject.
7. The method of claim 6, wherein said disease is an autoimmune
disease or allergic disease selected from the group comprising
multiple sclerosis, type 1 diabetes, rheumatoid arthritis, asthma,
chronic obstructive pulmonary diseases, inflammatory bowel disease,
Alzheimer's disease and other neurodegenerative diseases with
inflammatory components, atherosclerosis, vasculitis, and
cancer.
8. The method of claim 6, wherein said composition further
comprises an inhibitor of PDE4, an inhibitor of PDE7 or both an
inhibitor of PDE4 and an inhibitor of PDE7.
9. The method of claim 6, wherein said disease is multiple
sclerosis and said inhibitor of PDE8 is dipyridamole or a
clinically effective derivative thereof.
10. A method of modulating an inflammatory response in a subject
comprising administering to a subject in need thereof a composition
comprising an inhibitor of PDE8 in an amount effective to modulate
lymphocyte chemotaxis in said subject.
11. The method of claim 10, wherein said composition further
comprises an inhibitor of PDE4, an inhibitor or PDE7 or both an
inhibitor of PDE4 and an inhibitor of PDE7.
12. The method of claim 10, wherein said inhibitor of PDE8 is
dipyridamole or a clinically effective derivative thereof.
13. A method of treating a disease associated with lymphocyte
chemotaxis, lymphocyte adhesion to endothelial cells, or lymphocyte
transendothelial migration comprising administering to a subject in
need thereof a composition comprising an inhibitor of PDE8 in an
amount effective to reduce lymphocyte chemotaxis, lymphocyte
adhesion to endothelial cells, or lymphocyte transendothelial
migration in said subject.
14. The method of claim 13, wherein the disease is selected from
the group comprising arthritis, diabetes, inflammatory bowel
diseases and metastatic cancer or a predisposition thereto.
15. The method of claim 13, wherein said composition further
comprises an inhibitor of PDE4, an inhibitor of PDE7 or both an
inhibitor of PDE4 and an inhibitor of PDE7.
16. The method of claim 13, wherein said inhibitor of PDE8 is
dipyridamole or a clinically effective derivative thereof.
17. A method of inhibiting migration of a metastatic cancer cell,
comprising contacting said metastatic cancer cell with a
composition that preferentially inhibits cyclic nucleotide
phosphodiesterase (PDE) 8.
18. The method of claim 17, wherein the metastatic cancer cell is
derived from epithelial tissue.
19. The method of claim 17, wherein said composition further
comprises an inhibitor of PDE4, an inhibitor of PDE7 or both an
inhibitor of PDE4 and an inhibitor of PDE7.
20. The method of claim 17, wherein said inhibitor of PDE8 is
dipyridamole or a clinically effective derivative thereof.
21. A method of treating or delaying the progression of a
metastatic cancer comprising administering to a subject in need
thereof a composition comprising an inhibitor of PDE8 in an amount
effective to reduce migration of metastatic cancer cells.
22. The method of claim 21, wherein the metastatic cancer is
derived from epithelial tissue.
23. The method of claim 21, wherein said composition further
comprises an inhibitor of PDE4, an inhibitor of PDE7 or both an
inhibitor of PDE4 and an inhibitor of PDE7.
23. The method of claim 21, wherein said inhibitor of PDE8 is
dipyridamole or a clinically effective derivative thereof.
24. A method of reducing the severity of an inflammatory condition,
comprising administering to a subject suffering from or at risk of
developing said condition a synergistic combination of a PDE4
inhibitor and a PDE8 inhibitor.
25. A method of identifying a selective PDE8 inhibitor composition,
comprising contacting an activated lymphocyte with a candidate PDE
inhibitory compound and detecting migration of said activated
lymphocyte, wherein a reduction in migration of said activated
lymphocyte compared to an unstimulated lymphocyte indicates that
said candidate compound selectively inhibits a PDE8-mediated
inflammation.
26. A method of identifying a PDE8 inhibitor composition,
comprising contacting an activated lymphocyte with a candidate PDE
inhibitory compound and detecting migration of said activated
lymphocyte, wherein a reduction in migration of said activated
lymphocyte in the presence of said compound compared to in the
absence of the compound indicates that said candidate compound
inhibits a PDE8-mediated inflammation.
27. A method of identifying a selective PDE8 inhibitor composition,
comprising contacting an activated lymphocyte with a candidate PDE
inhibitory compound and detecting expression of activity of PDE8 in
said activated lymphocyte, wherein a reduction in said PDE 8
expression or activity compared to a PDE selected from the group
consisting of PDE1, 2, 3, 4, 5, 6, 7, 9, 10, or 11 indicates that
said candidate compound selectively inhibits a PDE8-mediated
inflammation.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/795,652, filed Apr. 27, 2006, the contents of
which are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to compositions and methods for
treating inflammation and other immune-related disorders.
BACKGROUND OF THE INVENTION
[0003] Inflammation is the complex biological response of vascular
tissues to harmful stimuli, such as pathogens, damaged cells, or
irritants.
[0004] Inflammation can be classified as either acute or chronic.
Acute inflammation is the initial response of the body to harmful
stimuli and is achieved by the increased movement of plasma and
leukocytes from the blood into the injured tissues. A cascade of
biochemical events propagates and matures the inflammatory
response, involving the local vascular system, the immune system,
and various cells within the injured tissue. Prolonged
inflammation, known as chronic inflammation, leads to a progressive
shift in the type of cells which are present at the site of
inflammation and is characterized by simultaneous destruction and
healing of the tissue from the inflammatory process.
[0005] Commonly, inflammation occurs as a defensive response to
invasion of the host by foreign, particularly microbial, material.
Responses to mechanical trauma, toxins, and neoplasia also may
results in inflammatory reactions. The accumulation and subsequent
activation of leukocytes are central events in the pathogenesis of
most forms of inflammation. Deficiencies of inflammation compromise
the host. Excessive inflammation caused by abnormal recognition of
host tissue as foreign or prolongation of the inflammatory process
may lead to inflammatory diseases as diverse as diabetes,
arteriosclerosis, cataracts, reperfusion injury, and cancer, to
post-infectious syndromes such as in infectious meningitis,
rheumatic fever, and to rheumatic diseases such as systemic lupus
erythematosus and rheumatoid arthritis. The centrality of the
inflammatory response in these varied disease processes makes its
regulation a major element in the prevention control or cure of
human disease.
[0006] Accordingly, there exists a need for methods of treating
inflammation and other immune-related disease.
SUMMARY OF THE INVENTION
[0007] The present invention comprises methods of treating diseases
of the immune system, particularly chronic diseases of the immune
system, and more particularly, autoimmune diseases such as multiple
sclerosis (MS). The invention features a method of inhibiting
migration of an activated lymphocyte to a site of inflammation by
contacting the lymphocyte with a composition that inhibits cyclic
nucleotide phosphodiesterase (PDE) 8. The composition inhibits
activated lymphocytes as well as unstimulated or unactivated
lymphocytes. The composition preferentially inhibits PDE8 compared
to other cellular PDEs such as PDE 1, 2, 3, 4, 5, 6, 7, 9, 10, or
11. For example, the inhibitor reduces PDE activity or expression
by at least 10%, 25%, 50%, 100%, 3-fold, 5-fold, 10-fold or more
compared to another PDE such as PDE4. The activated lymphocyte or
population of lymphocytes contains activated cells of the T helper
type 1 phenotype and/or T helper 17 phenotype. An example of a
preferential PDE8 inhibitor is dipyridamole or an derivative
thereof. As defined herein, the term "derivative", refers to
compounds that have a common core structure, and are substituted
with any of a variety of substituents. Other suitable PDE8A
inhibitors for use in the anti-inflammatory composition and methods
provided herein include PDE8A inhibitors such as E-4021 and
papaverine (Soderling, et al., Proc Natl Acad Sci USA, vol. 95:
8991-8996 (1998); and Gamanuma et al., Cellular Signalling, vol.
15:565-574 (2003)). Optionally, the composition also contains an
inhibitor of PDE4.
[0008] Also within the invention is an anti-inflammatory
composition containing a combination of a PDE8 inhibitor and a PDE4
inhibitor or a PDE8 inhibitor and a PDE7 inhibitor. The combination
of a PDE8 inhibitor and a second PDE inhibitor such as a PDE4
inhibitor or PDE7 inhibitor is a synergistic combination. The
combination optionally includes inhibitors of PDE8, PDE4, and PDE7.
Exemplary PDE4 inhibitors include Cilomilast (Ariflo, SB 207499,
SmithKline Beecham), Roflumilast, PLX 369, PLX 743 (Plexxikon,
Inc.),
N-(3,5-Dichloro-pyrid-4-yl)-[1-(4-fluorobenzyl)-5-hydroxy-indole-3-yl]-gl-
yoxylic acid amide (AWD 12-281), mesembrine (an alkaloid present in
the herb, Sceletium tortuosum), and rolipram. However, their
clinical use is limited due to adverse side effects. Exemplary PDE7
inhibitors include BRL 50481
[3-(N,N-dimethylsulfonamido)-4-methyl-nitrobenzene] (Smith et al.,
Mol. Pharmacol., vol. 66(6): 1679-1689 (2004)); IC242 (Lee et al.,
Cell Signal, vol. 14:277-284 (2002)); T-2585 (Nakata, et al., Clin
Exp Immunol., vol. 128(3): 460-6 (2002)), YM-393059 (Yamamoto et
al., Eur J. Pharmacol., vol. 541(1-2):106-14 (2006); and Yamamoto
et al., Eur J. Pharmacol. Vol. 559(2-3): 219-26 (2007)), a series
of compounds such as BMS-586353 described in Lorthiois et al.,
Bioorg. Med. Chem. Lett., vol. 14: 4623-26 (2004); Pitts et al.,
Bioorg. Med. Chem. Lett., vol. 14: 2955-58 (2004) and Vergne et
al., Bioorg. Med. Chem. Lett., vol. 14:4607-13 (2004)); and
8-bromo-9-substituted derivatives of guanine (Barnes et al.,
Bioorg. Med. Chem. Lett., vol. 14:1081-83 (2001)). The
co-administration compositions include, for example, compounds that
are dual PDE4 and PDE7 inhibitors, such as, for example, T-2585 and
YM-393059
[0009] Co-administration of a PDE4 inhibitor (and/or a PDE7
inhibitor) and a PDE8 inhibitor provides a synergistic
anti-inflammatory effect compared to administration of PDE4 (or
PDE7) alone or PDE8 alone. The combination is more efficacious with
the added advantage of reduced adverse side effects associated with
anti-inflammatory amounts of PDE4 alone. The combination optionally
includes other anti-inflammatory agents such as inhibitors of other
PDEs or any of a variety of known anti-inflammatory agents such
that the efficacy of the known anti-inflammatory agent is enhanced
by the combination and/or the therapeutically effective amount of
the known anti-inflammatory agent is reduced when used in
combination with a PDE inhibitor of the invention. Suitable
anti-inflammatory agents for use in such combination therapy
include, for example, glucocorticoids, anti-adhesion antibodies
such as anti-alpha 4 integrin antibodies, e.g., Tysabri,
interferons, and antagonists of TNF-alpha such anti-TNF-alpha
antibodies, e.g., Humira, Remicade and Enbrel. The synergistic
combination is more effective in inhibiting lymphocyte migration or
adhesion and reducing inflammation than either ingredient
alone.
[0010] A method of reducing or preventing a symptom associated with
a disorder of the immune system is carried out by administering to
a subject in need thereof a composition containing an inhibitor of
PDE8 in an amount effective to modulate, e.g., reduce, lymphocyte
chemotaxis. The methods described herein are useful for
administration to humans as well as other animals (e.g., dogs,
cats, horses, cattle, sheep, pigs) that are identified as suffering
from or at risk of developing an autoimmune or inflammatory
condition/disorder. For example, the disease state is selected from
the group comprising multiple sclerosis, rheumatoid arthritis,
asthma, and inflammatory bowel disease. Optionally, the composition
further contains an inhibitor of PDE4. The compounds are formulated
together in one composition or are formulated individually. The
PDE8 and PDE4 inhibitory compounds are co-administered or
administered in sequence, i.e., administration of one inhibitor
occurs before or after administration of the other inhibitor. The
compositions are administered systemically, e.g., orally,
intravenously, intramuscularly, or locally, e.g., topically, to a
site of inflamed tissue.
[0011] In one example, the disease is multiple sclerosis, an
autoimmune disease characterized by inflammation and an immune
response directed against myelin of nerve fibers, brain or spinal
cord. The inhibitor of PDE8 is dipyridamole or a clinically
effective derivative thereof. Inhibitory compounds are administered
during an episode to reduce pain and neurological symptoms or
compositions are administered during a period of remission to
prevent the occurrence of an episode and/or to reduce the severity
of a subsequent episode of multiple sclerosis.
[0012] The compositions and methods are useful to modulate, e.g.,
reduce an inflammatory response, in a subject. The PDE8 or PDE8/4
inhibitory compositions are administered to a subject in need
thereof in an amount effective to modulate, e.g., reduce,
lymphocyte chemotaxis in the subject thereby treating or
ameliorating the symptoms of a disease associated with lymphocyte
chemotaxis, lymphocyte adhesion to endothelial cells, and/or
lymphocyte transendothelial migration. Thus, the compounds are
administered in an amount effective to modulate, e.g., reduce,
lymphocyte chemotaxis, lymphocyte adhesion to endothelial cells,
and/or lymphocyte transendothelial migration in subject. Exemplary
disorders to be treated also include an autoimmune disease or
allergic disease selected from the group comprising multiple
sclerosis, type 1 diabetes, rheumatoid arthritis, asthma, chronic
obstructive pulmonary diseases, inflammatory bowel disease,
Alzheimer's disease and other neurodegenerative diseases with
inflammatory components, atherosclerosis, vasculitis, and cancer,
such as metastatic cancers. Other disorders or conditions to be
treated include inflammatory diseases and conditions such as joint
inflammation, rheumatoid arthritis, rheumatoid spondylitis,
osteoarthritis, chronic glomerulonephritis, dermatitis, and
inflammatory bowel disease, such as, for example, ulcerative
colitis and/or Crohn's disease; respiratory diseases and conditions
such as asthma, acute respiratory distress syndrome, chronic
pulmonary inflammatory disease, bronchitis, chronic obstructive
airway disease, and silicosis; infectious diseases and conditions
such as sepsis, septic shock, endotoxic shock, gram negative,
sepsis, toxic shock syndrome, fever and myalgias due to bacterial,
viral or fungal infection, and influenza; Alzheimer's disease and
other neurodegenerative diseases with an inflammatory component
immune diseases and conditions such as autoimmune diabetes,
systemic lupus erythematosis, graft vs. host reaction, allograft
rejections, multiple sclerosis, psoriasis, and allergic rhinitis.
Administration of PDE8 inhibitors or a synergistic combination of
PDE8/4 inhibitors also confer clinical benefit to those suffering
from other diseases and conditions such as bone resorption
diseases; reperfusion injury; cachexia secondary to infection or
malignancy; cachexia secondary to human acquired immune deficiency
syndrome (AIDS), human immunodeficiency virus (HIV) infection, or
AIDS related complex (ARC); keloid formation; scar tissue
formation; type 1 diabetes mellitus; or leukemia.
[0013] The compositions and methods are useful in treating,
reducing the spread of, delay the progression of or otherwise
preventing, migration of metastatic cancer cells. Thus, the
selective PDE inhibitors identified herein are useful in
modulating, e.g., reducing the migration of metastatic cancer
cells. Compositions useful in methods of reducing the migration of
metastatic cancer cells are delivered systematically by
administering these compositions to a subject in need thereof.
These compositions, for example, are formulated for oral
administration. Compositions useful in methods of treating or
alleviating a symptom of a metastatic cancer are administered, for
example, as an adjunct therapy in addition to other known
chemotherapy agents.
[0014] For example, the invention provides methods of inhibiting
migration of a metastatic cancer cell by contacting the metastatic
cancer cell with a composition that preferentially inhibits cyclic
nucleotide phosphodiesterase (PDE) 8. For example, the metastatic
cancer cell is derived from epithelial tissue. The composition
includes, for example, a second agent such as an inhibitor of PDE4,
an inhibitor of PDE7 or both an inhibitor of PDE4 and an inhibitor
of PDE7. The inhibitor of PDE8 is, for example, dipyridamole or a
clinically effective derivative thereof.
[0015] The invention also provides methods of treating or delaying
the progression of a metastatic cancer by administering to a
subject in need thereof a composition that includes an inhibitor of
PDE8 in an amount effective to reduce migration of metastatic
cancer cells. For example, the metastatic cancer cell is derived
from epithelial tissue. The composition includes, for example, a
second agent such as an inhibitor of PDE4, an inhibitor of PDE7 or
both an inhibitor of PDE4 and an inhibitor of PDE7. The inhibitor
of PDE8 is, for example, dipyridamole or a clinically effective
derivative thereof.
[0016] The methods include a step of diagnosing and/or identifying
a subject comprising a primary tumor selected from the group
consisting of colorectal, stomach, pancreatic, biliary tree, small
intestine, kidney, breast, prostate, ovarian, malignant melanoma,
lung cancer, and lymphoma. These cells of these primary tumor types
are characterized by metastatic migration mediated by chemokines
such as CXCL12.
[0017] The subset of cancers known as "metastatic cancers" refer to
those types of cancers in which the primary tumor is prone to
spread from its original site to another part of the subject, a
process known as metastasizing. In metastatic cancers, the primary
tumor is often an epithelial-derived tumor. Epithelial derived
tumor types that are prone to metastasizing include primary tumors
originating in the colon and rectum, stomach, pancreas, biliary
tree, small intestine, kidney, breast, prostate, ovaries, malignant
melanoma, lung cancer, and lymphoma. The compositions and methods
are useful in treating and/or preventing these metastatic cancers
by inhibiting or otherwise reducing the migration of the metastatic
cancer cells from the original site of the primary tumor to a
second site within a subject.
[0018] Methods of identifying and/or diagnosing an individual who
is suffering from or is at risk of developing a metastatic cancer
are known in the art. For example, detection of a serum marker
associated with metastatic cancer in an individual indicates that
the individual is, has, or is at risk of developing metastases. CT
scan or ultrasound is also used to confirm the presence of a tumor
in any of the tissues and organs listed above. Diagnosis of any one
of the above-listed primary tumors indicates that an individual is
at risk of developing a metastasis. Multiple metastatic lesions are
often the case, but single metastases may be seen. Optionally,
biopsy is carried out to confirm metastatic cancer.
[0019] The compositions and methods are also useful in treating,
alleviating a symptom of, delaying the progression of or otherwise
preventing cancers that are induced by chronic inflammation. For
example, the compounds and methods are useful in the treatment of a
carcinoma. These compositions and methods are useful in the
treatment of a cancer derived from epithelial tissues. For example,
the compositions and methods are useful in the treatment of colon
cancer and/or liver cancer. Compositions useful in methods of
treating or alleviating a symptom of cancers induced by chronic
inflammation are administered, for example, as an adjunct therapy
in addition to other known chemotherapy agents.
[0020] The methods are also useful in reducing the symptoms of
respiratory disorders such as allergen-induced or
inflammation-induced bronchial disorders such as bronchitis,
obstructive bronchitis, spastic bronchitis, allergic bronchitis,
allergic asthma, bronchial asthma, and chronic obstructive
pulmonary disease (COPD) as well as inflammatory conditions of the
gastrointestinal tract or bowel.
[0021] Those of ordinary skill in the art will appreciate that the
dosages and formulations for the administration of a selective PDE
inhibitor, alone or in combination with other selective PDE
inhibitors, is determined based on the variables such as the
potency of the inhibitor, the formulation, and the route of
administration. The dosages used in the methods and co-therapy
methods do not exceed the dosage at which the PDE inhibitor ceases
to be a selective for a given PDE gene family. For example,
Rolipram, the prototypical PDE4 inhibitor, will for example lose
its specificity above 10 .mu.M.
[0022] In the methods provided herein, the selective PDE8 inhibitor
is administered at a dosage in the range of 0.01 to 100 mg/kg/day.
The PDE8 inhibitor is administered at a dosage in the range of 0.1
to 10 mg/kg/day. For example, the PDE8 inhibitor is administered at
dosage selected from 5 mg/kg/day, 3 mg/kg/day, 2.5 mg/kg/day, 2
mg/kg/day, 1.5 mg/kg/day, 1 mg/kg/day, 0.75 mg/kg/day and 0.5
mg/kg/day.
[0023] In the methods provided herein, the dosage of the PDE4
inhibitor depends on the inhibitor used. For example, roflumilast
is administered at a dosage in the range of 100 .mu.g/day to 1
mg/day, and preferably in the range of 250 .mu.g/day to 500
.mu.g/day. For example, roflumilast is administered once daily at a
dosage of 500 .mu.g/day. PDE4 inhibitors such as cilomilast are
administered at a dosage between 1 mg/kg/day and 100 mg/kg/day and
preferably at a dosage between 10 mg/kg/day and 30 mg/kg/day. For
example, PDE4 inhibitors such as cilomilast are administered twice
daily at a dosage in the range of 5 mg/kg to 15 mg/kg. (Lipworth,
Lancet, vol. 365:167-75 (2005)).
[0024] A further object of the present invention is to treat
diseases of the immune system at the level of regulating and/or
controlling the migration and subsequent action, such as adhesion,
of activated lymphocytes. The invention also comprises methods of
regulating and/or controlling, primarily methods of inhibiting, the
recruitment of activated lymphocytes to sites of inflammation to
thereby decrease the severity and/or duration of such inflammation.
Inflammatory disorders include, for example, chronic and acute
inflammatory disorders.
[0025] The invention comprises the use of inhibitors of one or more
cyclic nucleotide phosphodiesterases (PDE) to reduce or block the
migration of activated lymphocytes, and thereby reduce the
subsequent adhesion and/or infiltration of such lymphocytes. The
PDEs comprise a family of related enzymes encoded by at least 21
different genes, grouped into 11 different gene families (PDEs # 1
to 11).
[0026] Of the PDEs, PDE4, PDE7 and PDE8 are known to be present in
mouse and human lymphocytes. Certain of these PDEs, such as PDE4,
have been shown to be induced during lymphocyte activation. It has
further been shown that inhibition of PDE4 alone is not sufficient
to effectively block the migration of such activated
lymphocytes.
[0027] The present invention demonstrates that inhibition of PDE8
is also needed in order to block migration and subsequent actions
of activated lymphocytes. The invention further demonstrates in a
clinical experiment in mammals that inhibition of PDE8 results in a
profound decrease in the observed symptoms, such as paralytic
signs, in a model mammalian system for experimental autoimmune
encephalitis (EAE). It is a further object of the present invention
to use methods for inhibiting PDE8 as methods of treating
inflammatory autoimmune or allergic diseases such as multiple
sclerosis, rheumatoid arthritis, asthma and inflammatory bowel
disease.
[0028] The invention provides methods of modulating, e.g.,
treating, reducing, alleviating or otherwise preventing,
inflammation or other immune-related diseases by administering
selective PDE inhibitors, and preferably, at least a selective PDE8
inhibitor. The selective PDE8 inhibitor is, for example, a
commercially available selective inhibitor, such as dipyridamole.
Other suitable selective PDE8 inhibitors include compounds
identified by screening chemical libraries for novel compounds that
inhibit PDE8 with the same or better ability as dipyridamole. Other
suitable selective PDE8 inhibitors are created, for example,
through rational design. In one embodiment, the selective PDE8
inhibitor is rationally designed to have a structure that is based
on the structure of dipyridamole or a derivative thereof.
[0029] Selective inhibitors of PDE8 are administered alone or in
combination with other suitable therapeutic agents. For example,
the selective PDE8 inhibitor is administered in combination with
one or more additional PDE inhibitors, such as, a PDE4
inhibitor.
[0030] The invention provides methods of modulating, e.g.,
treating, reducing, alleviating or otherwise preventing,
inflammation or other immune-related diseases by administering a
selective inhibitor that targets a novel PDE isoform that is
identified using the methods provided herein. Suitable selective
PDE inhibitors include, for example, a commercially available
selective inhibitor, compounds identified by screening known
chemical libraries for novel compounds that inhibit the novel PDE
isoform, and inhibitors created through rational design.
[0031] Targeting PDE8 and other novel PDE isoforms maximizes the
therapeutic potential in the treatment of inflammation, while
simultaneously increasing the therapeutic index of PDE inhibition.
Targeting of these PDE isoforms therefore overcomes the limitations
observed in human anti-inflammatory therapies with selective PDE4
inhibitors.
[0032] The selective PDE inhibitors used in the methods of the
invention, such as, the selective PDE8 inhibitors, are administered
in an amount that is effective to treat, reduce, alleviate or
otherwise prevent multiple sclerosis and other autoimmune diseases
associated with chemokine-induced migration of leukocytes.
[0033] In addition to targeting pro-inflammatory T effector cells,
the selective PDE inhibitors are also useful in treating,
alleviating, delaying the progression of, or otherwise preventing
inflammatory diseases through immune deviation or induction of
immunosuppressive regulatory T cells (Tregs) in vivo. Studies have
shown that signals required to induce and maintain Tregs include
Foxp3-dependent repression of PDE3B (Gavin, et al., Nature,
ePublication, (Jan. 14, 2007)). A PDE inhibitor such as a PDE8
inhibitor contributes to regulatory T cell development and
function, and is, therefore, effective in the treatment of
inflammatory diseases (Qiao, et al., Immunology, vol. 120(4):447-55
(2007); Shevach et al., Immunol Rev 212:60-73 (2006).
[0034] The invention also provides methods of identifying putative
selective PDE inhibitors, such as, for example, PDE8 inhibitors by
determining the ability of a test compound to inhibit the migration
of stimulated and unstimulated splenocytes, for example, using the
chemotaxis assays disclosed herein. Screening methods to identify
anti-inflammatory compositions to inhibit migration and/or adhesion
of activated lymphocytes are carried out as follows. A method of
identifying a selective PDE8 inhibitor composition is carried out
by contacting an activated lymphocyte with a candidate PDE
inhibitory compound and detecting migration of the activated
lymphocyte. The assay is carried out using a population of
stimulated lymphocytes or a mixed population of stimulated and
unstimulated cells. Optionally, the cells are genetically modified,
e.g., the cells lack expression of one or more PDEs (e.g., cells
from a PDE-/- knockout mouse). A reduction in migration of an
activated lymphocyte compared to migration of an unstimulated
lymphocyte indicates that the candidate compound selectively
inhibits a PDE8-mediated anti-inflammation.
[0035] Another method of identifying a selective PDE8 inhibitor
composition includes the following steps: contacting an activated
lymphocyte with a candidate PDE inhibitory compound and detecting
migration of the activated lymphocyte, wherein a reduction in
migration of the activated lymphocyte in the presence of the
compound compared to in the absence of the compound indicates that
the candidate compound inhibits a PDE8-mediated inflammation.
[0036] Another method of identifying a selective PDE8 inhibitor
composition includes the following steps: contacting an activated
lymphocyte with a candidate PDE inhibitory compound and detecting
expression or activity of PDE8 in the activated lymphocyte. A
reduction in PDE 8 expression or activity compared to a PDE
selected from the group consisting of PDE1, 2, 3, 4, 5, 6, 7, 9,
10, or 11 indicates that the candidate compound selectively
inhibits a PDE8-mediated anti-inflammation.
[0037] The invention also provides methods of identifying putative
selective PDE inhibitors, such as PDE8 inhibitors, by determining
the ability of a test compound to inhibit one or more effector
functions of an inflammatory cell population. For example, putative
PDE inhibitors are identified by their ability to inhibit
proliferation, cytotoxicity and/or cytokine production such as the
production of interleukin-2, interferon gamma, interleukin-17
and/or TNF-alpha. The level of effector function in the presence or
absence of a test compound is identified, for example, using
standard assays including ELISA assays.
[0038] Other features, objects, and advantages of the invention
will be apparent from the description and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 is a graph depicting the effect of CXCL12 on
migration of splenocytes.
[0040] FIG. 2 is a graph depicting the effect of dibutyryl cAMP on
splenocyte chemotaxis in response to CXCL12.
[0041] FIG. 3 is a graph depicting the effect of forskolin and IBMX
on splenocyte chemotaxis in response to CXCL12.
[0042] FIG. 4 is a graph depicting the effect of PDE gene
family-specific inhibitors on chemotaxis of stimulated splenocytes
in response to CXCL12.
[0043] FIG. 5 is a graph depicting the effect of dipyridamole on
splenocyte chemotaxis in response to CXCL12.
[0044] FIGS. 6A and 6B are a series of graphs depicting the effect
of Rp-cAMPS and adenosine deaminase on dipyridamole inhibition of
splenocyte chemotaxis in response to CXCL12.
[0045] FIG. 7 is a graph depicting the expression of mRNA for
PDE8A1, PDE4B2 and PDE7A1 following Con A stimulation.
[0046] FIG. 8 is a graph depicting the effect of dipyridamole (DP)
treatment on experimental autoimmune encephalomyelitis (EAE).
[0047] FIG. 9 is an illustration depicting the role of PDEs in
regulation of signal transduction.
[0048] FIG. 10 is a graph depicting the effect of PDEs isoforms on
the regulation of cAMP/PKA signaling in T cells.
[0049] FIGS. 11A and 11B are a series of graphs depicting the
effect of PDE8 on splenocyte adhesion to a bEND.3 monolayer. In
FIG. 11A, Con A activated splenocytes were adhered to bEND.3 cells
in the presence of dipyridamole (100 .mu.M) or piclamast (1
.mu.M).
[0050] FIG. 12 is a graph depicting the suppression of inflammation
by dipyridamole.
[0051] FIG. 13 is an illustration depicting the experimental scheme
for the identification of novel PDE targets in stimulated T cells
treated with selective PDE inhibitors.
[0052] FIG. 14 is an illustration depicting the experimental scheme
for determining regulatory functions of novel PDE isoforms in
anti-CD3 and anti-CD28 stimulated cells and T cells treated with
selective PDE inhibitors.
[0053] FIG. 15 is an illustration depicting recruitment steps I-IV
(I, rolling; II, adhesion; III, locomotion; IV, transendothelial
migration (TEM)) as measured in the flow chamber assay by real-time
videomicroscopy.
[0054] FIG. 16 is an illustration depicting the experimental scheme
for analyzing the individual and overlapping functions of PDE1,
PDE3, PDE4, PDE7 and PDE8 gene families during inflammation.
[0055] FIG. 17 is an illustration depicting the experimental scheme
for determining the effect of PDE7 inhibitors and/or PDE8
inhibitors on the dosage requirements of PDE4 inhibitors in an EAE
model in vivo.
[0056] FIG. 18 is a schematic illustration of the PDE8A1
protein.
DETAILED DESCRIPTION
[0057] The methods provided herein employ PDE inhibitor therapy to
modulate, e.g., reduce, inhibit, treat or prevent inflammatory
illnesses and other immune-related diseases. The immune system
depends on chemokines to recruit lymphocytes to tissues in
inflammatory diseases. Thus, the invention provides methods of
modulating inflammatory diseases by inhibiting migration of
activated lymphocytes using selective PDE inhibitors. The Examples
provided herein identified phosphodiesterase 8 (PDE8) as a target
for inhibition of chemotaxis of activated lymphocytes. In
particular, chemotactic responses of unstimulated and concanavalin
A-stimulated mouse splenocytes and their modulation by agents that
stimulate the cAMP signaling pathway were compared. Dibutyryl cAMP
inhibited migration of both cell types. In contrast, forskolin and
3-isobutyl-1-methylxanthine each inhibited migration of
unstimulated splenocytes, with little effect on migration of
stimulated splenocytes. Dipyridamole alone, a PDE inhibitor capable
of inhibiting PDE8, strongly inhibited migration of stimulated and
unstimulated splenocytes, and this inhibition was enhanced by
forskolin and reversed by a PKA antagonist. Following concanavalin
A stimulation, mRNA for PDE8A1 was induced.
[0058] Thus, the methods provided herein modulate, e.g., inhibit
migration of activated lymphocytes using selective inhibitors of
PDE8. The invention also provides methods of identifying other PDE
isoforms as targets for PDE inhibitor therapy. Thus, the methods
provided herein also include methods of modulating, e.g., reducing,
inhibiting, treating or preventing inflammatory illnesses and other
immune-related diseases by administering inhibitors of these
identified PDE isoforms to reduce or block the migration of
activated lymphocytes, and thereby reducing the subsequent adhesion
and/or infiltration of such lymphocytes.
[0059] Phosphodiesterases (PDEs) are a family of related enzymes
codes by at least 21 different genes, grouped into 11 different
gene families (PDEs # 1 to 11). The data provided herein
demonstrated that cyclic nucleotide phosphodiesterases (PDEs)
expressed in activated T cells contribute to the control of
effector T cell functions and thereby serve, in concert with PDE4,
as targets for the treatment of inflammation. T cell functions and
inflammation are tested in mice in which the genes for each of the
PDEs present in activated T cells are deleted or inhibited
pharmacologically through the use of selective PDE inhibitors both
in vitro and in vivo.
[0060] Extravasation of T cells in post-capillary venules plays an
important role in the pathogenesis of various inflammatory
conditions. Thus, uncovering a means of inhibiting T cell
recruitment and effector functions as described herein provided the
basis for an effective treatment of inflammatory illnesses.
Observations that PDE4 is the most abundantly expressed form of PDE
in T cells, and that inhibition of PDE4 blocks T cell activation
and function through elevating cAMP, generated considerable
interest in developing pharmacological inhibitors of PDE4 as
potential therapies for treatment of chronic inflammatory diseases,
including chronic obstructive pulmonary disease (COPD). It is now
accepted that individual PDE isoforms serve to modulate distinct
regulatory pathways in cells, including T cells (Conti and Beavo.,
Annu Rev Biochem., ePublication (Mar. 21, 2007)). These properties
therefore offer the opportunity for selectively targeting specific
PDEs for treatment of specific disease states. However, despite
high expectations for PDE4 inhibitors to treat inflammatory
illnesses, when used in clinical trials, PDE4 inhibitors were less
efficacious than preclinical data suggested; consequently, none has
been approved for clinical use. Further, PDE4 inhibitors exhibit a
low therapeutic index due to dose-limiting side effects which
hampered their clinical development (Burnouf and Pruniaux, Curr
Pharm Des, vol. 8:1255-1296 (2002); Giembycz, Proc Am Thorac Soc,
vol. 2:326-333; discussion 340-321 (2005); Giembycz, Curr Opin
Pharmacol, vol. 5:238-244 (2005); Giembycz and Smith, Curr Pharm
Des, vol. 12:3207-3220 (2006); and Bender and Beavo. Pharmacol Rev,
vol. 58:488-520 (2006)). Recent reports have shown roles for PDEs
other than PDE4 in controlling cAMP signaling in T cells. It was
reported that PDE7 and possibly PDE8 are required for T cell
activation and induced by CD3 and CD28 stimulation (Li et al.,
Science, vol. 283:848-851 (1999); and Glavas et al., Proc Natl Acad
Sci USA, vol. 98:6319-6324 (2001)). Therefore, PDE4 inhibitors may
have shown limited efficacy in clinical trials because important
PDE isoforms induced in activated T cells were not targeted. The
studies described herein identified PDE8 as a novel target for
inhibition of T cell chemotaxis. The Examples provided herein also
provide studies that are designed to translate this approach to
effector T cells and inflammation as a prelude to developing
comprehensive PDE inhibitor treatments for inflammatory
disorders.
[0061] With this in mind, it was hypothesized that 1) selective
isoforms of the PDE1, PDE3, PDE7, and PDE8 gene families are
induced in activated T cells causing inflammation, 2) as a
consequence of this, in addition to members of the PDE4 gene family
that are constitutively expressed in T cells, these induced
isoforms are critical regulators of T cell functions, and 3) in
order to maximize the effectiveness of PDE inhibition for full
control of accumulation of activated T cells in tissues, it is
necessary to identify and inhibit PDE isoforms in addition to PDE4
that are important in controlling T cell function.
[0062] The studies are carried out to identify novel PDE targets in
anti-CD3 and anti-CD28 stimulated T cells treated with selective
PDE inhibitors and from wildtype and PDE mutant mice.
Identification of these targets is accomplished by first analyzing
in vitro the expression of isoforms of the PDE1, PDE3, PDE4, PDE7
and PDE8 gene families in T cells using quantitative real-time
RT-PCR (qRT-PCR) and Western immunoblotting procedures. This is
done in anti-CD3 and anti-CD28 stimulated T cells from wildtype
(wt) as well as specific PDE gene knock out (PDE.sup.-/-) mice and
selective PDE inhibitor-treated T cells to detect compensatory
changes in PDE isoform expression during T cell activation.
[0063] Studies are also carried to determine the regulatory
functions of novel PDE isoforms in anti-CD3 and anti-CD28
stimulated T cells from wildtype and PDE mutant mice and T cells
treated with selective PDE inhibitors. Functions of defined PDEs in
regulating T cell activity are determined by testing T cells in
proliferation and cytokine production assays. The role of members
of the PDE1, PDE3, PDE4, PDE7 and PDE8 gene families in regulating
cAMP-PKA-dependent vascular T cell recruitment is then determined
by real time videomicroscopy measuring rolling and arrest,
activation and adhesion strengthening, and transendothelial
migration (TEM) under physiologic shear stress in vitro.
[0064] Unique and overlapping functions of PDE1, PDE3, PDE4, PDE7
and PDE8 gene families during inflammation in vivo are determined.
To accomplish this, the susceptibility to experimental inflammation
of wt mice is compared to specific PDE.sup.-/- mice. Additionally,
the therapeutic effect of pharmacologic inhibition of specific PDE
gene families is tested using selective PDE inhibitors alone or in
combination during experimental inflammation in vivo.
cAMP and T cell function
[0065] Studies in vitro and in vivo have shown that T cell
proliferation as well as effector and regulatory functions can be
modulated by cAMP (Conti and Beavo, Annu Rev Biochem., ePublication
(Mar. 21, 2007); Giembycz, Curr Opin Pharmacol, vol. 5:238-244
(2005); Bender and Beavo. Pharmacol Rev, vol. 58:488-520 (2006);
Asirvatham, et al., J Immunol, vol. 173:4806-4814 (2004); Gavin, et
al., Nature, ePublication, (Jan. 14, 2007); vol. 445(7129):771-5
(2007); and Conti, et al. J Biol Chem, vol. 278:5493-5496 (2003).
To be fully activated, CD4+ T cells need engagement of the T cell
receptor (TCR)-CD3 complex and costimulation through molecules such
as CD28. cAMP inhibits T cell function (Laudanna, et al., J Biol
Chem, vol. 272:24141-24144 (1997)) and proliferation by exerting
negative control on the TCR and costimulatory receptor pathways (Li
et al., Science, vol. 283:848-851 (1999); and Glavas et al., Proc
Natl Acad Sci USA, vol. 98:6319-6324 (2001)). Complete T cell
activation is believed to result from a reduction in cAMP,
releasing the negative hold (Conti and Beavo, Annu Rev Biochem.,
ePublication (Mar. 21, 2007) and Bender and Beavo. Pharmacol Rev,
vol. 58:488-520 (2006)).
PDEs as Regulators of cAMP Signaling and Therapeutic Targeting
[0066] The second messenger cAMP plays important roles in mediating
the biological effects of a wide variety of first messengers
(Bender and Beavo. Pharmacol Rev, vol. 58:488-520 (2006); and
Lerner and Epstein. Biochem J, vol. 393:21-41 (2006)). Increases in
intracellular cAMP lead to activation of cAMP-dependent protein
kinases, guanine nucleotide exchange factors, and cyclic
nucleotide-gated channels, which in turn can regulate the activity
of other signaling and metabolic pathways (FIG. 9). In the model of
the second-messenger concept originally put forth by Sutherland and
colleagues (shown in FIG. 9), first messengers, such as hormones,
neurotransmitters, cytokines and growth factors, upon interacting
with receptors on the cell surface, generate the production of a
`second messenger` such as cAMP, which then redirects the machinery
of the cell, affecting many physiological processes. Currently,
three different types of effector proteins to which cAMP can bind
and carry out its actions are known: PKA (cAMP-dependent protein
kinase), EPAC (exchange protein activated by cAMP) and CNGCs
(cyclic nucleotide-gated channels). Activation of PKA by cAMP leads
to changes both in cytosolic proteins and in gene transcription
through phosphorylation of cAMP-responsive nuclear factors such as
CREB, CREM (CRE modulator) and ATF-1 (activating transcription
factor 1). PDEs, by regulating cAMP levels, play a central role in
modulating all of these cAMP signalling pathways and consequent
physiological responses. AC, adenylate cyclase; R, receptor
(seven-membrane-spanning G-protein-coupled metabotropic receptor);
Gs, G protein coupled to stimulation of adenylyl cyclase.
[0067] cAMP signaling pathways are controlled through regulation of
the synthesis of cAMP by adenylyl cyclases and degradation by PDEs.
The cyclic nucleotide-PDEs are now recognized to form a superfamily
of 11 different, but homologous, gene-families that all contain
highly homologous catalytic domains near their C termini and give
rise to more than 50 protein products (Bender and Beavo. Pharmacol
Rev, vol. 58:488-520 (2006); Lerner and Epstein. Biochem J, vol.
393:21-41 (2006); Soderling and Beavo. Curr Opin Cell Biol, vol.
12:174-179 (2000); and Francis, et al., Prog Nucleic Acid Res Mol
Biol, vol. 65:1-52 (2001)). PDE-catalyzed cyclic nucleotide
degradation provides an important mechanism for regulating
signaling. Inhibition of PDEs reduces cAMP hydrolysis resulting in
subsequent elevations in cAMP levels within the cell. Given the
large number of different PDE isoforms now known to exist, and
their selective expression and localization within cells and
subcellular regions, it is now recognized that inhibition of
different forms of PDE results in increases in cAMP in restricted
microdomains within the cell (Mongillo, et al. Circ Res, vol.
95:67-75 (2004); Rich, et al., Proc Natl Acad Sci USA, vol.
98:13049-13054 (2001); and Rich, et al., J Gen Physiol, vol.
118:63-78 (2001)). Indeed, it is now acknowledged that the PDE
component of cAMP pathways ensures the proper intensity and
spatiotemporal distribution of the signal, as illustrated by many
studies on different endocrine tissues (Conti, et al. J Biol Chem,
vol. 278:5493-5496 (2003); and Conti, Mol Endocrinol, vol.
14:1317-1327 (2000)).
Targeting PDEs
[0068] At the time of the discovery of PDE activity, it was also
found that caffeine (1,3,7-trimethylxanthine), which is
structurally similar to the substrate, cAMP, was an effective
competitive inhibitor of PDE activity and a number of nonselective
PDE inhibitors including the caffeine analog, theophylline
(1,3-dimethylxanthine), have been in use as therapeutic agents for
asthma and several other illnesses for decades (Bender and Beavo.
Pharmacol Rev, vol. 58:488-520 (2006)). Thus, the principle that
PDEs are valid targets and that inhibition of PDE activity is a
valid therapeutic approach to treatment of inflammatory diseases is
now well accepted. However, since the early PDE inhibitors were
non-selective and targeted most known PDE isoforms, they had a very
narrow therapeutic index. Nevertheless, one important reason that
PDEs have been pursued as therapeutic targets is related to the
basic pharmacological principle that regulation of degradation of
any ligand or second messenger can often make a more rapid and
larger percentage change in concentration than comparable
regulation of the rates of synthesis. This is true for either
pharmacokinetic changes in drug levels or changes in amounts of an
endogenous cellular regulatory molecule or metabolite. Indeed, it
has been found that almost all tissues contain at least an order of
magnitude higher maximal PDE activity than cyclase activity for
either cAMP or cGMP (Conti, et al. J Biol Chem, vol. 278:5493-5496
(2003)). It has been apparent for many years now that there is a
rather extraordinarily large number of different forms of PDEs
expressed in mammalian tissues, each of which can have a unique
architecture at the active site. Moreover, there is increasing
evidence that many of these PDEs are tightly connected to different
physiological functions in the body and by inference also to
different pathological conditions. Therefore, it has been widely
believed that it should be possible to develop isoform selective
inhibitors that can target specific functions and pathological
conditions without a high likelihood of causing nonspecific side
effects. The recent therapeutic and commercial success of agents
such as sildenafil (Viagra), a selective PDE5 inhibitor, has
validated the concept.
PDEs and T Cells
[0069] It is well established that PDE3 (3B) and PDE4 (4A,B and D)
constitute the major gene families and activity in T cells (Tenor,
et al., Clin Exp Allergy, vol. 25:616-624 (1995); and Giembycz, et
al., Br J Pharmacol, vol. 118:1945-1958 (1996)). However, recent
evidence suggests that additional isoforms, including PDE1 (1B1,
1B2), PDE7 (7A1, 7A3), and PDE8 (8A1) are expressed and upregulated
in T cells as a response to activation signals (Li et al., Science,
vol. 283:848-851 (1999); Glavas et al., Proc Natl Acad Sci USA,
vol. 98:6319-6324 (2001); Jiang, et al., Proc Natl Acad Sci USA,
vol. 93:11236-11241 (1996); and Bender, et al., Proc Natl Acad Sci
USA, vol. 102:497-502 (2005)). Thus, determining the spatiotemporal
distribution and functional significance of the entire spectrum of
PDEs expressed within effector/memory T cells remains a critical
challenge to be addressed.
[0070] cAMP has long been known to inhibit lymphocyte activation,
proliferation and function (Bourne, et al., Science, vol. 184:19-28
(1974); and Kammer, Immunol Today, vol. 9:222-229 (1988)). cAMP was
also reported to inhibit motility of cytotoxic T lymphocytes
(Valitutti, et al., Eur J Immunol, vol. 23: 790-795 (1993)).
Inasmuch as recruitment of lymphocytes to sites of inflammation,
i.e., their migration through postcapillary endothelial layers and
subsequent parenchymal accumulation (Rot and von Andrian, Annu Rev
Immunol, vol. 22: 891-928 (2004)) underlies the basis of a number
of autoimmune diseases, such as multiple sclerosis (Engelhardt, and
Ransohoff, Trends Immunol 26: 485-495 (2005); Steinman, Science
305: 212-216 (2004); and Steinman, Cell, vol. 85: 299-302 (1996)),
stimulating the cAMP signaling pathway in lymphocytes as a means to
inhibit their migration, has been suggested as the basis of very
effective treatments for these diseases (Bielekova, et al., J
Immunol, vol. 164:1117-1124 (2000)). cAMP levels in cells are
controlled by their synthesis by adenylyl cyclases and degradation
by cyclic nucleotide phosphodiesterases (PDEs). PDEs comprise a
superfamily of related enzymes encoded by at least 21 different
genes, grouped into 11 different gene families (PDEs 1-11), based
on sequence similarity, mode of regulation and preference for cAMP
or cGMP as substrate (Soderling, and Beavo, Curr Opin Cell Biol,
vol. 12: 174-179 (2000); Francis, et al., Prog Nucleic Acid Res Mol
Biol, vol. 65: 1-52 (2001); and Lerner, and Epstein, Biochem J,
vol. 393: 21-41 (2006)). Some are cAMP-selective hydrolases (PDE 4,
-7 and -8), others are cGMP-selective hydrolases (PDE 5, -6 and -9)
and the rest hydrolyze both cAMP and cGMP (PDE1, -2, -3, -10 and
-11). A review of the expression and function of the PDE family in
normal haematopoietic cells is found, e.g., in Lerner, and Epstein,
Biochem J, vol. 393: 21-41 (2006), the teachings of which are
hereby incorporated by reference in their entirety. Additional PDEs
are disclosed, for example, in U.S. Patent Application Publication
No. 2005/0058998 by Beavo et al., the teachings of which are hereby
incorporated by reference in their entirety.
[0071] The role of PDEs in controlling the cAMP signaling with
respect to T cell function is exemplified by a report by Beavo and
colleagues that showed PDE7 is required for T cell activation and
that PDE7 and PDE8 are induced by anti-CD3 and anti-CD28 (Li et
al., Science, vol. 283:848-851 (1999); and Glavas et al., Proc Natl
Acad Sci USA, vol. 98:6319-6324 (2001)). When cAMP is increased by
inhibiting PDE7A there is also less IL-2 production which leads to
less T cell proliferation (Li et al., Science, vol. 283:848-851
(1999)). A comprehensive review of the expression of PDEs in T
cells has recently been published (Lerner and Epstein. Biochem J,
vol. 393:21-41 (2006)). Initial studies have shown the predominant
activity in the cytosol of isolated human peripheral blood
lymphocytes to be comprised of high affinity cAMP-specific activity
with kinetic, catalytic and inhibitor specificity properties,
molecular mass and elution profile from anion exchange columns to
be representative of PDE4 (Epstein, et al., Adv Cyclic Nucleotide
Protein Phosphorylation Res, vol. 16:303-324 (1984)). Later, after
the PDE1-5 gene families were discovered and defined, expression
pattern of PDEs in highly purified populations of CD4+ and CD8+
human T cells were examined by activity analysis using
family-specific PDE inhibitors to assess the relative contributions
of the different PDE families known to exist at that time (FIG. 10)
(Tenor, et al., Clin Exp Allergy, vol. 25:616-624 (1995)). Both
CD4+ and CD8+ T cells showed identical PDE expression patterns,
with PDE4 comprising about 60% of the total activity and PDE3 about
25%. The majority of the PDE4 activity was cytosolic, whereas the
PDE3 activity was almost exclusively particulate. Only very low
amounts (.ltoreq.1-3%) of PDE1, PDE2 and PDE5 were expressed, and
about 15% of the total activity could not be assigned to PDEs 1-5,
owing to its insensitivity to specific inhibitors of these families
(Tenor, et al., Clin Exp Allergy, vol. 25:616-624 (1995)). Inasmuch
as the residual 15% unassigned activity exhibited a very low Km of
.apprxeq.0.05-0.08 .mu.M, it was theorized that this might
represent the then newly discovered PDE7, which possesses a Km for
cAMP in that range. Indeed, the PDE7A gene was subsequently shown
to be expressed in human T lymphocyte cell lines (Bloom and Beavo,
Proc Natl Acad Sci USA, vol. 93:14188-14192 (1996)) and in isolated
human peripheral blood T lymphocytes (Giembycz, et al., Br J
Pharmacol, vol. 118:1945-1958 (1996)).
[0072] Analyses of the expression patterns of PDEs in mice have not
been studied as extensively as that in humans, but where reported,
the expression patterns of PDEs in mice appear similar to that in
humans. A single report on PDEs in isolated mouse thymocytes was
published in which the PDE activity in the cytosol of these cells,
as analyzed by inhibitor sensitivities, was shown to consist of
about 80% PDE4 and about 20% PDE2 (Michie, et al. Cell Signal, vol.
8:97-110 (1996)). Shortly afterward the PDE7A gene was cloned from
mouse and it was shown to be 98% identical to the human PDE7A gene,
and the mouse PDE7A1 splice variant was shown to exhibit an
identical tissue expression pattern to that in human, with highest
expression in tissues of the immune system--thymus, spleen and
lymph nodes (Wang, et al., Biochem Biophys Res Commun, vol.
276:1271-1277 (2000)). A subsequent study on a PDE7A gene knockout
in mouse showed measurable PDE3, PDE4 and PDE7 activities in whole
cell lysates of lymphocytes isolated from mouse lymph nodes, with
activity levels of each of these PDEs similar to those seen in
humans (Yang, et al., J Immunol, vol. 171:6414-6420 (2003)). Thus
the PDE expression patterns in resting human and mouse T
lymphocytes appear to be nearly identical, with the possible
exception of more PDE2 expressed in mouse than in human, at least
based on one report.
[0073] In addition to the expression of PDE3, PDE4 and PDE7 in
resting lymphocytes, members of the PDE1 and PDE8 gene families are
found in activated cells. Whereas resting lymphocytes contain
little or no detectable PDE1, appreciable expression of the PDE1B
gene is seen in human leukemic cells (Epstein, et al., Biochem J,
vol. 243:533-539 (1987)) and mouse S49 lymphoma cells (Repaske, et
al., J Biol Chem, vol. 267:18683-18688 (1992)), and its expression
is induced in isolated human peripheral blood lymphocytes following
mitogenic stimulation (Jiang, et al., Proc Natl Acad Sci USA, vol.
93:11236-11241 (1996)). Further, cloning of the human form of PDE1B
showed it to be 96% identical to that from mouse (Jiang, et al.,
Proc Natl Acad Sci USA, vol. 93:11236-11241 (1996)). Following the
more recent discovery of the PDE8 gene family, the PDE8A gene was
also shown to be induced in both human (Glavas et al., Proc Natl
Acad Sci USA, vol. 98:6319-6324 (2001) and mouse (Dong, et al.,
Biochem Biophys Res Commun, vol. 345:713-719 (2006)) T cells
following mitogenic stimulation. Two splice variants of the PDE7A
gene, 7A1 and 7A3 are also induced following activation of human T
cells (Li et al., Science, vol. 283:848-851 (1999); and Glavas et
al., Proc Natl Acad Sci USA, vol. 98:6319-6324 (2001)). Activation
of lymphocytes also results in induction of members of the PDE4
gene family, PDE4A4, PDE4D1/D2, and PDE4D3 (Jiang, et al., Cell
Biochem Biophys, vol. 28:135-160 (1998)), as well as translocation
of PDE4A4, PDE4B2 and PDED1/D2 to lipid rafts (Abrahamsen et al., J
Immunol, vol. 173:4847-4858 (2004)). Of the three known PDE1 genes,
T cells express the PDE1B gene (Jiang, et al., Proc Natl Acad Sci
USA, vol. 93:11236-11241 (1996)); of the two known PDE3 genes they
express the PDE3B gene (Sheth, et al. Br J Haematol, vol.
99:784-789 (1997); and Seybold, et al., J Biol Chem, vol.
273:20575-20588 (1998)); of the four known PDE4 genes they express
the PDE4A, 4B and 4D genes (Giembycz, et al., Br J Pharmacol, vol.
118:1945-1958 (1996); Jiang, et al., Cell Biochem Biophys, vol.
28:135-160 (1998); and Seybold, et al., J Biol Chem, vol.
273:20575-20588 (1998)); of the two known PDE7 genes they express
the PDE7A gene (Giembycz, et al., Br J Pharmacol, vol.
118:1945-1958 (1996); and Yang, et al., J Immunol, vol.
171:6414-6420 (2003)); and of the two known PDE8 genes, they
express the PDE8A gene (Glavas et al., Proc Natl Acad Sci USA, vol.
98:6319-6324 (2001)).
Targeting of Mouse Lymphocyte PDEs as a Model for Targeting of
Human T Cell PDEs in the Treatment of Human Autoimmune Diseases
[0074] Mouse models employing specific PDE gene knockouts represent
excellent models for targeting these PDEs in human diseases because
1) PDEs are encoded by 21 different genes in humans, and mice
express these same 21 PDE genes 2) all 21 PDE genes contain a
catalytic domain of about 270 amino acids near their carboxyl
terminal end which exhibits 20-45% sequence homology among all of
these expressed gene products; however, for any given expressed PDE
gene, the sequence homology is >95% between mouse and human
species, and 3) as a result of this close sequence identity, family
specific PDE inhibitors that target human forms of PDE show the
exact same potency and specificity for the corresponding mouse PDEs
(Bender and Beavo, Pharmacol Rev, vol. 58:488-520 (2006); Francis,
et al., Prog Nucleic Acid Res Mol Biol, vol. 65:1-52 (2001); and
Conti and Jin, Prog Nucleic Acid Res Mol Biol, vol. 63:1-38
(1999)).
PDE Control of T Cell Functions During Inflammation in Mutant Mice
and Mice Treated with Selective Inhibitors
[0075] Over the last 15 years there has been considerable interest
in the cAMP-specific, or PDE4, family of enzymes as intracellular
targets that could be exploited to therapeutic advantage for a
multitude of diseases associated with chronic inflammation (Burnouf
and Pruniaux, Curr Pharm Des, vol. 8:1255-1296 (2002); Castro, et
al., Med Res Rev, vol. 25:229-244 (2005); Barnette, et al., J
Pharmacol Exp Ther, vol. 284:420-426 (1998); and Souness, et al.,
Immunopharmacology, vol. 47:127-162 (2000)). However, although drug
targeting of PDE4 is based on a conceptually robust hypothesis
(Giembycz, Proc Am Thorac Soc, vol. 2:326-333; discussion 340-321
(2005); Giembycz, Curr Opin Pharmacol, vol. 5:238-244 (2005);
Bender and Beavo, Pharmacol Rev, vol. 58:488-520 (2006);
Bernareggi, et al., Br J Pharmacol, vol. 128:327-336 (1999);
Giembycz, Br J Clin Pharmacol, vol. 62:138-152 (2006); Sommer et
al., J Neuroimmunol, vol. 79:54-61. (1997); Jung et al., J
Neuroimmunol, vol. 68:1-11 (1996); Moore et al., J Pharmacol Exp
Ther, vol. 319:63-72 (2006); Martinez et al., Brain Res, vol.
846:265-267 (1999); Bielekova, et al., J Immunol, vol.
164:1117-1124 (2000); Lagente et al., Mem Inst Oswaldo Cruz, vol.
100 Suppl 1:131-136 (2005); and Ouagued et al., Pulm Pharmacol
Ther, vol. 18:49-54 (2005)), dose-limiting side effects, of which
nausea and vomiting are the most common and troublesome, have
hampered their clinical development. A fundamental challenge that
still is to be met by the pharmaceutical industry is to synthesize
compounds with an improved therapeutic ratio given that the adverse
effects of PDE4 inhibitors represent an extension of their
pharmacology. Several strategies are being considered to dissociate
the beneficial from detrimental effects of PDE4 inhibitors with
some degree of success (Giembycz, Proc Am Thorac Soc, vol.
2:326-333; discussion 340-321 (2005); Giembycz, Curr Opin
Pharmacol, vol. 5:238-244 (2005); Bender and Beavo. Pharmacol Rev,
vol. 58:488-520 (2006); Bernareggi, et al., Br J Pharmacol, vol.
128:327-336 (1999); and Giembycz, Br J Clin Pharmacol, vol.
62:138-152 (2006)). However, compounds with an optimal
pharmacophore have not yet been reported. An alternative approach,
that is a subject of current research, is to inhibit other cAMP PDE
families that are expressed in immune and proinflammatory cells in
the hope that therapeutic activity can be retained at the expense
of side effects. The most promising candidates are PDE7 and PDE8.
This study was carried out to assess the contribution of each of
the seven PDE genes found to be expressed in T cells, PDEs 1B, 3B,
4A, 4B, 4D, 7A, and 8A, for their role in regulation of
inflammatory T cells functions, and as targets for the amelioration
of the clinical signs of experimental inflammation in vivo.
[0076] PDE4 is one of the major PDE gene families expressed in both
human (Epstein, and Hachisu, Adv Cyclic Nucleotide Protein
Phosphorylation Res, vol. 16: 303-324 (1984); Tenor, et al., Clin
Exp Allergy, vol. 25: 616-624 (1995); and Giembycz, et al., Br J
Pharmacol, vol. 118: 1945-1958 (1996)) and mouse (Michie, et al.,
Cell Signal, vol. 8: 97-110 (1996)) lymphocytes, accounting for
most of the hydrolysis of cAMP. With this observation in mind, the
PDE4-selective inhibitor, rolipram, was tested in previous studies
to determine its effect on lymphocyte chemotaxis. This
PDE4-selective inhibitor was found to inhibit lymphocyte migration
stimulated by platelet activating factor, interleukin-8, and CXCL12
(stromal cell-derived factor-1) (Hidi, et al., Eur Respir J, vol.
15: 342-349 (2000); and Layseca-Espinosa, et al., J Invest
Dermatol, vol. 121: 81-87 (2003)).
[0077] These in vitro studies showing effects of rolipram on
lymphocyte migration were done with unstimulated, quiescent
lymphocytes, but it is widely accepted that the population of
lymphocytes that migrate to the site of inflammation and across the
endothelium in vivo mostly belong to previously activated
lymphocyte subsets and represent activated cells with a T helper
type I phenotype (Nourshargh, and Marelli-Berg, Trends Immunol,
vol. 26: 157-165 (2005)). Moreover, it is also widely known that
the expression profile and localizations of PDEs in activated
lymphocytes differ from that in unstimulated cells. Indeed, early
studies had shown a long term induction of 5-10-fold in lymphocyte
PDE activity following stimulation by phytohaemagglutinin (Epstein,
et al., Cancer Res, vol. 40: 379-386 (1980)) or concanavalin A (Con
A) (Epstein, et al., Cancer Res, vol. 40: 379-386 (1980); and
Takemoto, et al., Biochem Biophys Res Commun, vol. 90: 491-497
(1979)). In addition, subsequent studies reported the long term
induction in activated lymphocytes of a variety of specific PDE
forms, such as PDE1B1 (Jiang, et al., Proc Natl Acad Sci USA, vol.
93 11236-11241 (1996)), PDE1B2 (Bender, et al., Proc Natl Acad Sci
USA, vol. 102: 497-502 (2005)), PDE4A4 (Jiang, et al., Cell Biochem
Biophys, vol. 28: 135-160 (1998)), PDE4D1/D2 (Jiang, et al., Cell
Biochem Biophys, vol. 28: 135-160 (1998)), PDE4D3 (Jiang, et al.,
Cell Biochem Biophys, vol. 28: 135-160 (1998)), PDE7A1 (Li, et al.,
Science, vol. 283: 848-851 (1999)), PDE7A3 (Glavas, et al., Proc
Natl Acad Sci USA, vol. 98 6319-6324 (2001)) and PDE8A1 (Glavas, et
al., Proc Natl Acad Sci USA, vol. 98 6319-6324 (2001)).
Furthermore, studies have also reported the translocation of
PDE4A4, PDE4B2 and PDE4D1/D2 to lipid rafts following lymphocyte
activation (Abrahamsen, et al., J Immunol, vol. 173: 4847-4858
(2004)).
[0078] Given these changes in PDEs that occur following lymphocyte
activation, it was hypothesized that the modulation of chemotactic
responses of activated lymphocytes by agents that stimulate the
cAMP signaling pathway could be quite different from that of
unstimulated lymphocytes. The studies disclosed herein were carried
out to examine this hypothesis. The modulation of the chemotactic
response exhibited by unstimulated and Con A-stimulated splenocytes
in the presence of agents that stimulate the cAMP signaling pathway
was compared. Whereas the cell permeable cAMP analogue, dibutyryl
cAMP readily inhibited chemotaxis of both cell populations,
surprisingly, the adenylyl cyclase activator, forskolin, and the
general non-selective PDE inhibitor, IBMX, inhibited migration of
unstimulated splenocytes, but either had no effect at all
(forskolin) or only a limited effect (IBMX) on activated cells,
except when added together. Since direct addition of an analogue of
cAMP inhibits migration of both cell types, whereas broad
modulators of the synthetic and degradative enzymes that regulate
cAMP primarily inhibited migration only of unstimulated cells,
unstimulated and activated splenocytes must differ in the way in
which they regulate cAMP within the cell.
[0079] Stimulated splenocytes, like unstimulated splenocytes,
responded to the chemokine CXCL12, a powerful chemoattractant for
leukocytes (Rot and von Andrian, Annu Rev Immunol, vol. 22: 891-928
(2004)), and stimulated splenocytes were readily inhibited in their
migration by the cAMP analogue, dibutyryl cAMP; however, unlike
unstimulated cells, migration of stimulated splenocytes was not
significantly affected by activators of adenylyl cyclase or
inhibitors of cAMP PDEs, except for dipyridamole, which differs
from the other PDE inhibitors used only in its ability to inhibit
PDE8. Further, quantitative real-time RTPCR revealed an induction
of PDE8 mRNA in splenocytes following Con A stimulation. The
studies disclosed herein demonstrate that, for activated
lymphocytes, the major therapeutic target in inflammatory
autoimmune diseases, inhibition of PDE4 is not sufficient to block
their recruitment into sites of inflammation. Thus, the methods
provided herein inhibit PDE8 to achieve a full therapeutic
response.
[0080] It has become increasingly apparent in recent years that in
response to stimuli, cAMP elevations in cells occur in a directed
spatial and temporal manner, resulting in the formation of
microdomains of localized cAMP concentrations within the cell, and
it is the regulation of these localized domains of cAMP in
physiologically important compartments that regulate specific
functions of the cell (Rich, et al., Proc Natl Acad Sci USA, vol.
98: 13049-13054 (2001)). It is now established that one way
microdomains of localized cAMP concentrations are achieved in the
cell is through selective expression and compartmentalization of
different isoforms of PDE (Rich, et al., J Gen Physiol, vol. 118:
63-78 (2001); Brunton, Sci STKE 2003: PE44 (2003); Mongillo, et
al., Circ Res, vol. 95: 67-75 (2004); and Baillie, et al., FEBS
Lett, vol. 579: 3264-3270 (2005)). Since migration of unstimulated
splenocytes was readily inhibited by the general non-selective PDE
inhibitor, IBMX, and activated splenocytes were far less affected
by IBMX, it was hypothesized that activated splenocytes might
express an IBMX-insensitive PDE activity in a functionally relevant
cell compartment linked to regulation of cell migration, which
limits the accumulation of cAMP in that compartment in response to
activators of adenylyl cyclase, IBMX or PDE selective inhibitors
targeted to IBMX-sensitive PDE gene families. Indeed, in addition
to the relative insensitivity to IBMX, migration of activated
splenocytes was also found to be resistant to inhibition by gene
family specific inhibitors targeted to PDE3, PDE4, and PDE7. The
inhibition of stimulated splenocytes seen following combined
addition of forskolin and IBMX could result from excessive
increases in cAMP causing spillover from one cellular compartment
to another, and overwhelming the normal cAMP degradative system in
the compartment responsible for regulation of migration.
[0081] Inasmuch as PDE8 is the only known cAMP hydrolyzing PDE gene
family reported to be resistant to IBMX inhibition, studies were
carried out to evaluate whether high expression of PDE8 in
activated splenocytes confers resistance of these cells to
inhibition of migration by adenylyl cyclase activators and PDE
inhibitors. If true, then inhibition of PDE8 in activated
splenocytes should inhibit their chemotactic migration.
Pharmacological characterization of expressed forms of PDE8 showed
them to be resistant to inhibition by all known PDE inhibitors
tested against them, with the exception of the non-selective PDE
inhibitor, dipyridamole, which inhibits PDE8A with reported IC50s
of 4-9 .mu.M (Soderling, et al., Proc Natl Acad Sci USA, vol. 95:
8991-8996 (1998); Fisher, et al., Biochem Biophys Res Commun, vol.
246: 570-577 (1998); and Gamanuma, et al., Cell Signal, vol. 15:
565-574 (2003)).
[0082] The structure of dipyridamole is as follows:
##STR00001##
[0083] Dipyridamole was then tested in the chemotactic assay system
described in the Examples below. In support of the hypothesis, the
studies disclosed herein demonstrated that dipyridamole profoundly
inhibited chemotactic migration of both unstimulated and stimulated
splenocytes.
[0084] Moreover, when forskolin was added along with dipyridamole,
to stimulate adenylyl cyclase, inhibition of migration was
potentiated, leading to as much as =70-80% inhibition of migration
of both unstimulated and stimulated cells. The structure of
forskolin is as follows:
##STR00002##
[0085] That dipyridamole is working through a cAMP mediated effect
is borne out by the reversal of the dipyridamole effect that is
seen with the protein kinase A (PKA) antagonist, Rp-cAMPS.
[0086] In a further test of the hypothesis, quantitative real-time
RT-PCR was performed to assess the mRNA expression for PDE4B2,
PDE7A1, and PDE8A1, following Con A stimulation of splenocytes.
Consistent with the hypothesis, a 2.7-fold induction of mRNA for
PDE8A1 was found, with no increase in mRNA for PDE 7A1, and only a
transient 1.5-fold increase in mRNA for PDE4B2. Hence, it is
conceivable that this increase in the expression of PDE8A in
activated splenocytes is responsible for conferring relative
resistance of these cells to inhibition of chemotactic migration by
adenylyl cyclase activators and PDE inhibitors other than
dipyridamole.
[0087] Studies have been run to determine why lymphocytes induce
PDE8 in a cell compartment functionally linked to regulation of
migration following cell activation. Nearly 30 years ago studies
reported 10-20-fold increases in PDE activity in proliferating and
transformed lymphocytes as compared to quiescent cells, and a
5-10-fold, long term induction of PDE activity in human peripheral
blood lymphocytes following mitogenic stimulation. Based on these
findings, it was postulated that induction of PDE might be
important to the mitogenic process (Epstein, et al., Cancer Res,
vol. 40: 379-386 (1980); and Epstein, et al., Cancer Res, vol. 37:
4016-4023 (1977)). It was then hypothesized that the increased PDE
activity might serve as a protective mechanism to ensure that cAMP
levels are not elevated, from circulating hormones and other
activators of adenylyl cyclase such as adenosine, to the point of
being inhibitory to activated lymphocytes, thereby ensuring that
the cells can traverse the stages necessary for commitment to
mitogenesis. Subsequent studies have largely borne this out and
have shown that PDEs 1 and 4, both of which are induced, are to a
large extent, responsible for controlling cAMP levels linked to
proliferation following lymphocyte activation (Jiang, et al., Proc
Natl Acad Sci USA, vol. 93 11236-11241 (1996); Jiang, et al., Cell
Biochem Biophys, vol. 28: 135-160 (1998); and Kanda, and Watanabe,
Biochem Pharmacol, vol. 62: 495-507 (2001)). A similar hypothesis
is tested herein; that is, that the induction of PDE8, in part,
functions to protect the activated lymphocytes from being inhibited
in their migration by agents in the circulation that can stimulate
adenylyl cyclase, and elevate cAMP in a compartment that can affect
migration.
[0088] Previous studies had reported induction of PDE7A1 in human T
lymphocytes activated with anti-CD3/CD28 antibodies, and the
inhibition of T cell proliferation by antisense inhibition of
PDE7A1 expression (Li, et al., Science, vol. 283: 848-851 (1999)).
It was therefore surprising to see no change in PDE7A1 mRNA in
mouse splenocytes following their activation. However, the
complement of PDEs in human and mouse T lymphocytes differ. For
example, whereas mice express PDE2 as one of the major PDE forms in
T cells (Michie, et al., Cell Signal, vol. 8: 97-110 (1996)), PDE2
is not expressed at all in human T or B cells (Tenor, et al., Clin
Exp Allergy, vol. 25: 616-624 (1995) and Gantner, et al., Br J
Pharmacol, vol. 123: 1031-1038 (1998)). Further, T lymphocyte
activation and cytokine production were completely normal in PDE7A
knockout mice, suggesting that in contrast to humans, PDE7A may
have little or no functional role in murine T cells (Yang, et al. J
Immunol, vol. 171: 6414-6420 (2003)). In contrast, similar to what
is shown in the Examples below in mice, PDE8A mRNA was upregulated
following activation of human T cells, and was hypothesized to play
an important role in the activation process, suggesting that PDE8
plays similar functionally important roles in both human and murine
lymphocytes (Glavas, et al., Proc Natl Acad Sci USA, vol. 98
6319-6324 (2001)).
[0089] Dipyridamole has other actions in addition to its inhibition
of PDEs, and it is possible that its inhibition of migration of
activated splenocytes may be due to another action. Dipyridamole is
a potent inhibitor of adenosine uptake into cells, and is used
clinically, under the trade name Persantine, for this action during
pharmacological stress tests. Since adenosine deaminase had no
effect on migration of splenocytes and did not attenuate the
dipyridamole inhibition of migration, it suggests that dipyridamole
is not acting in this system through inhibition of adenosine
uptake. However, even though dipyridamole may act to elevate
extracellular adenosine, the inhibitory actions of extracellular
adenosine on lymphocyte proliferation and function are nevertheless
believed to result from elevation of intracellular cAMP, as a
result of stimulation of adenylyl cyclase through purinergic
receptors (DosReis, et al., Cell Immunol, vol. 101: 213-231
(1986)). Hence, dipyridamole is thought to stimulate the cAMP
signaling pathway in two ways, through indirect stimulation of
adenylyl cyclase as a result of elevation of adenosine, and through
direct inhibition of PDEs. Dipyridamole has anti-inflammatory
properties and was recently reported to attenuate nuclear
translocation of NF.kappa.B and block the synthesis of monocyte
chemoattractant protein-1 in platelet-monocyte aggregates (Weyrich,
et al., Circulation, vol. 111: 633-642 (2005)). Much of the
anti-inflammatory properties of dipyridamole results from its
ability to inhibit chemotactic migration of activated lymphocytes,
as shown in the study provided herein.
[0090] During inflammation, leukocytes respond to chemokines
presented at the luminal surface of the endothelium through their
G-protein coupled high affinity receptors and migrate into
underlying tissues. Since chemokine-induced transendothelial
migration of activated T lymphocytes is one of the first steps of
the pathological process in chronic inflammatory autoimmune
diseases such as multiple sclerosis, blockage of this migration
should provide an effective therapeutic means of treating
autoimmune diseases (Steinman, Science 305: 212-216 (2004) and
Steinman, Nat Rev Drug Discov, vol. 4: 510-518 (2005)). A review of
the pathophysiology of autoimmune diseases and targeted immune
therapies for autoimmune diseases is provided, e.g., in Steinman,
Science 305: 212-216 (2004), the teachings of which are hereby
incorporated by reference in their entirety.
[0091] The studies provided herein show that PDE8 is a critical
target for inhibiting the migration of activated lymphocytes. The
methods of modulating, e.g., treating, reducing, alleviating or
otherwise preventing, inflammation or other immune-related diseases
employ selective PDE inhibitors, and preferably, at least a
selective PDE8 inhibitor. The selective PDE8 inhibitor is, for
example, a commercially available selective inhibitor, such as
dipyridamole. Other suitable selective PDE8 inhibitors include
compounds identified by screening known chemical libraries for
novel compounds that inhibit PDE8 with the same or better ability
as dipyridamole. Other suitable selective PDE8 inhibitors are
created using strategies such as rational design, for example,
based on the structure of dipyridamole or a derivative thereof.
Putative selective PDE8 inhibitors are screened and/or tested for
their ability to inhibit the migration of stimulated and
unstimulated splenocytes, for example, using the chemotaxis assays
disclosed herein.
[0092] All mammalian PDEs identified to date contain a catalytic
region comprised of approximately 270 amino acids located toward
the C-terminus. The different PDEs share between about 20-45%
identity in this region. Crystal structures have been determined
for the catalytic sites of PDEs 1, 3, 4, 5, 7 and 9, but not yet
for PDE8. Development of PDE8 inhibitors is based on the principles
of binding of substrate and inhibitors to the catalytic site
derived from these solved crystal structures. In particular, since
PDE8 and PDE4 are strictly cAMP-specific, information gathered from
binding to the active site of PDE4 is used in the development of
PDE8 inhibitors.
[0093] From all the PDE crystal structures solved so far, the
catalytic domains are composed of 16 alpha helices consisting of
three sub-domains that define a deep pocket where substrate or
inhibitors bind. The active site pocket contains 11 of the 17
conserved residues in all PDEs. Important residues are contributed
by each of the 3 sub-domains. Two divalent metal binding sites,
both involved in catalysis, are found at the bottom of the
substrate binding pocket; one site for a tightly bound Zn++ and
another for a loosely bound Mg++. The Zn++ binding site has two
histidine and two aspartic acid residues that are conserved among
all known PDEs. All structures show an invariant glutamine that
stabilizes the binding of the purine ring. For dual specificity
PDEs, this glutamine must be able to rotate freely to form hydrogen
bonds with both cAMP and cGMP. For PDEs that are specific for cAMP,
this glutamine is constrained by neighboring residues into a
favored orientation for cAMP, and similarly, for cGMP-specific
PDEs, it is constrained into a different position that favors cGMP.
As PDE8 and PDE4 are both cAMP-specific, inhibitors of PDE8 are
based on structural determinants of dipyridamole, E4021 and
papaverine, which have some ability to inhibit PDE8, and the known
residues important for substrate and inhibitor binding of PDE4.
[0094] The crystal structure of the catalytic domain of PDE4B, the
first to be solved, and a model for the binding of cAMP substrate
to PDE4B was proposed. The preferred model for cAMP binding to the
catalytic pocket of PDE4B is the anti conformation of cAMP where
the adenine base is inserted into the lipophilic pocket by Leu393,
Pro396, Ile410, Phe414, and Phe446. The cyclic phosphate group
binds to the two metal binding sites. The 1-N and 6-NH.sub.2 groups
form hydrogen bonds with the side chain of the invariant Gln443,
while the 7-N position forms a more distorted hydrogen bond with
asn395. The ribose ring binds loosely against Met347 and Leu393,
with a hydrogen bond between His234 and the O3' oxygen, but with no
obvious interaction with the O2', O4', and O5' atoms.
[0095] Interaction of PDEs with scaffold proteins, which target
them to specific subcellular regions, is critical for correct
signaling to occur. Hence, interference of the specific binding of
PDEs to their scaffold proteins disrupts their function. PDE8 is
the only PDE gene family that contains a PAS (Per-Arnt-Sim) domain
through which it most likely binds to other proteins. Indeed, a
PAS-dependent physical association of PDE8A1 with endogenous
IkappaB has been shown by antibody array. Binding of PDE8A1
competes with the p65/p50 NF-kappaB for IkappaB binding, and the
binding of IkappaB to PDE8A increases its catalytic activity
6-fold. Therefore, inhibitors are designed to target to the PAS
region of PDE8A to disrupt the binding of PDE8A to other partner or
scaffold proteins, as a means to further block the function of this
enzyme.
[0096] The PDE8 inhibitors are designed to bind to at least a
portion the catalytic domain of the PDE8A1 isoform so as the
interfere with the interaction between PDE8A1 and cAMP. Preferably,
these PDE8 inhibitor competes with cAMP for binding to the active
site of PDE8A1. A diagram of the PDE8A1 protein is shown in FIG.
18. These PDE8 inhibitors are designed to bind to the catalytic
domain region of PDE8A1, which is located between amino acid
residues 555-797 of the amino acid sequence shown in GenBank
Accession No. NP.sub.--002596, or the corresponding amino acid
residues encoded by the PDE8A1 nucleic acid sequences in GenBank
Accession Nos. NM.sub.--002605, BC060762; BC075822 and
NM173454.
[0097] Other suitable PDE8 inhibitors are designed to bind to a
portion of the PDE8A1 isoform that is separate and distinct from
the catalytic domain, wherein that portion of PDE8A1 is a binding
site for a secondary molecule, such as a chaperone protein. For
example, PDE8 inhibitors are designed to bind to at least a portion
of the PAS region of PDE8A1 so as the interfere with the
interaction between PDE8A1 and a chaperone protein, such as, for
example, I kappa beta. Preferably, these PDE8 inhibitor competes
with I kappa .beta. for binding to the PAS region of PDE8A1. These
PDE8 inhibitors are designed to bind to the PAS region of PDE8A1,
which is located between amino acid residues 215-281 of the amino
acid sequence shown in GenBank Accession No. NP.sub.--002596, or
the corresponding amino acid residues encoded by the PDE8A1 nucleic
acid sequences in GenBank Accession Nos. NM.sub.--002605, BC060762;
BC075822 and NM173454.
[0098] Selective inhibitors of PDE8 are administered alone or in
combination with other suitable therapeutic agents. For example,
the selective PDE8 inhibitor is administered in combination with
one or more additional PDE inhibitors, such as, a PDE4 inhibitor
(Bielekova, et al., J Immunol, vol. 164:1117-1124 (2000)), a PDE7
inhibitor (Li et al., Science 283:848-851 (1999); and Glavas et
al., Proc Natl Acad Sci USA 98:6319-6324 (2001)) or both a PDE4
inhibitor and a PDE7 inhibitor.
[0099] The selective PDE inhibitors used in the methods of the
invention, such as, the selective PDE8 inhibitors, are administered
in an amount that is effective to treat, reduce, alleviate or
otherwise prevent multiple sclerosis and other autoimmune diseases
associated with chemokine-induced migration of leukocytes. For
example, the selective PDE inhibitors are used in an amount that is
effective to treat, reduce, alleviate, delay the progression of or
otherwise prevent an autoimmune disease or allergic disease
selected from the group comprising multiple sclerosis, type 1
diabetes, rheumatoid arthritis, asthma, chronic obstructive
pulmonary diseases, inflammatory bowel disease, Alzheimer's disease
and other neurodegenerative diseases with inflammatory components,
atherosclerosis, vasculitis, and cancer.
[0100] As used herein, the term "antagonist" or "inhibitor" refers
to a molecule which, when bound to a PDE, decreases the amount
(i.e., expression) or the duration of the effect of the biological
or immunological activity of the PDE. Antagonists may include small
molecules, proteins, polypeptides, peptides, nucleic acids,
carbohydrates, antibodies or any other molecules which decrease the
amount (expression) or effect of PDEs present in the sample. The
preferred antagonist selectively inhibits the biological activity
of a PDE, while not affecting any other cellular proteins.
[0101] As used herein, the term "modulates" refers to a change in
the activity of PDEs. For example, modulation may cause an increase
or a decrease in protein amount or activity, binding
characteristics, or any other biological, functional or
immunological properties of PDEs.
[0102] As used herein, the term "biological sample" is used in its
broadest sense. A biological sample is suspected of containing
nucleic acid encoding PDEs, or fragments thereof, or a PDE protein
itself or fragments thereof. The suitable biological sample is
from, e.g., an animal or a human. The sample is a cell sample or a
tissue sample, including samples from spleen, lymph node, thymus,
bone marrow, liver, heart, testis, brain, placenta, lung, skeletal
muscle, kidney and pancreas. The sample is a biological fluid,
including, urine, blood sera, blood plasma, phlegm, or lavage
fluid. Alternatively, the sample is a swab from the nose, ear or
throat.
[0103] The term "T cell activation" as used herein refers to a
process by which T cells change from a resting state to one where
they are proliferating and producing interleukins. In vivo, T cell
activation occurs when an antigen-presenting cell (APC) binds to
the T cell via the T cell receptor/CD3 complex and another
co-stimulatory molecule, such as CD28. In vitro, T cell induction
can be induced by binding anti-mouse antibodies beads to a plate.
When antibodies to murine anti-CD3 and anti-CD28 antibodies are
added to the plate, they bind to the anti-mouse antibodies by their
Fc regions. This leaves the Fab region free to bind CD3 and CD28
receptors on T cells. When T cells are added to the plate, they
bind to the antibodies attached to the bottom of the plate and
become activated, resulting in T cell proliferation and production
of interleukins. The plate with attached antibodies approximates an
APC which has receptors that bind to CD3 and CD28.
[0104] A variety of abbreviations are used throughout. "cAMP" is an
abbreviation for cyclic AMP or cyclic 3',5'-adenosine
monophosphate. cAMP is a small nucleotide regulatory molecule that
functions to regulate many fundamental cell processes. "cGMP" is
cyclic 3',5'-guanosine monophosphate, another regulatory molecule
that can be hydrolyzed by some forms of PDE. "COPD" refers to
chronic obstructive pulmonary disease. "EAE" refers to experimental
autoimmune encephalomyelitis, a T cell-mediated inflammatory
disease of the CNS that serves as an animal model of the human
disease multiple sclerosis. "GAP" refers to GTPase activating
protein which accelerates the intrinsic GTP-hydrolytic activity of
RhoA to produce the GDP-bound inactive state. "GEF" refers to
guanine nucleotide exchange factor which promotes GDP release and
GTP binding to RhoA. "GDI" refers to guanine nucleotide
dissociation inhibitor which sequesters RhoA in its GDP bound state
and interferes with its membrane association. "mAb" refers to
monoclonal antibody. "PDE" refers to an abbreviation for cyclic
nucleotide phosphodiesterase(s). These are the enzymes that
terminate the actions of cAMP by breaking it down to 5'-AMP, and
they are thus predominantly responsible for controlling the levels
of cAMP in cells. "PKA" refers to cAMP-dependent protein kinase.
"qRT-PCR" refers to quantitative real-time reverse
transcriptase-polymerase chain reaction. "RhoA" refers to a small
GTP binding protein that possesses GTPase activity belonging to the
ras superfamily of GTP binding proteins. "ROCK" refers to a protein
kinase that is activated by RhoA. "Rp-cAMPS" refers to 3',5'-cyclic
monophosphorothioate, Rp-isomer. This is a stereoisomer analogue of
cAMP that acts as an antagonist of PKA by competing for cAMP for
binding to the PKA regulatory subunits.
Pharmaceutical Compositions
[0105] The compounds of the invention can be useful in the
prevention or treatment of a variety of human or other animal,
including mammalian and non mammalian, disorders, including
primarily inflammatory disorders and other immune-related diseases.
It is contemplated that, once identified, the active molecules of
the invention, such as the selective PDE inhibitor, preferably at
least a selective PDE8 inhibitor, can be incorporated into any
suitable carrier prior to use. The dose of active molecule, mode of
administration and use of suitable carrier will depend upon the
intended recipient and target disorder. The formulations, both for
veterinary and for human medical use, of inhibitors according to
the present invention typically include such inhibitors in
association with a pharmaceutically acceptable carrier.
[0106] The carrier(s) should be "acceptable" in the sense of being
compatible with the other ingredients of the formulations and not
deleterious to the recipient. Pharmaceutically acceptable carriers,
in this regard, are intended to include any and all solvents,
dispersion media, coatings, anti-bacterial and anti-fungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. The use of such media and
agents for pharmaceutically active substances is known in the art.
Except insofar as any conventional media or agent is incompatible
with the active compound, use thereof in the compositions is
contemplated. Supplementary active compounds (identified or
designed according to the invention and/or known in the art) also
can be incorporated into the compositions. The formulations can
conveniently be presented in dosage unit form and can be prepared
by any of the methods well known in the art of
pharmacy/microbiology. In general, some formulations are prepared
by bringing the inhibitors into association with a liquid carrier
or a finely divided solid carrier or both, and then, if necessary,
shaping the product into the desired formulation.
[0107] A pharmaceutical composition of the invention should be
formulated to be compatible with its intended route of
administration. Examples of routes of administration include oral
or parenteral, for example, intravenous, intradermal, inhalation,
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide.
[0108] Useful solutions for oral or parenteral administration can
be prepared by any of the methods well known in the pharmaceutical
art, described, for example, in Remington's Pharmaceutical
Sciences, (Gennaro, A., ed.), Mack Pub., (1990). The parenteral
preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic. Suppositories for
rectal administration also can be prepared by mixing the drug with
a non-irritating excipient such as cocoa butter, other glycerides,
or other compositions which are solid at room temperature and
liquid at body temperatures. Formulations also can include, for
example, polyalkylene glycols such as polyethylene glycol, oils of
vegetable origin, and hydrogenated naphthalenes. Formulations for
direct administration can include glycerol and other compositions
of high viscosity. Other potentially useful parenteral carriers for
these drugs include ethylene-vinyl acetate copolymer particles,
osmotic pumps, implantable infusion systems, and liposomes.
Formulations for inhalation administration can contain as
excipients, for example, lactose, or can be aqueous solutions
containing, for example, polyoxyethylene-9-lauryl ether,
glycocholate and deoxycholate, or oily solutions for administration
in the form of nasal drops, or as a gel to be applied intranasally.
Retention enemas also can be used for rectal delivery.
[0109] Formulations of the present invention suitable for oral
administration can be in the form of: discrete units such as
capsules, gelatin capsules, sachets, tablets, troches, or lozenges,
each containing a predetermined amount of the drug; a powder or
granular composition; a solution or a suspension in an aqueous
liquid or non-aqueous liquid; or an oil-in-water emulsion or a
water-in-oil emulsion. The drug can also be administered in the
form of a bolus, electuary or paste. A tablet can be made by
compressing or molding the drug optionally with one or more
accessory ingredients. Compressed tablets can be prepared by
compressing, in a suitable machine, the drug in a free-flowing form
such as a powder or granules, optionally mixed by a binder,
lubricant, inert diluent, surface active or dispersing agent.
Molded tablets can be made by molding, in a suitable machine, a
mixture of the powdered drug and suitable carrier moistened with an
inert liquid diluent.
[0110] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients. Oral
compositions prepared using a fluid carrier for use as a mouthwash
include the compound in the fluid carrier and are applied orally
and swished and expectorated or swallowed. Pharmaceutically
compatible binding agents, and/or adjuvant materials can be
included as part of the composition. The tablets, pills, capsules,
troches and the like can contain any of the following ingredients,
or compounds of a similar nature: a binder such as microcrystalline
cellulose, gum tragacanth or gelatin; an excipient such as starch
or lactose; a disintegrating agent such as alginic acid, Primogel,
or corn starch; a lubricant such as magnesium stearate or Sterotes;
a glidant such as colloidal silicon dioxide; a sweetening agent
such as sucrose or saccharin; or a flavoring agent such as
peppermint, methyl salicylate, or orange flavoring.
[0111] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). It should be stable under the
conditions of manufacture and storage and should be preserved
against the contaminating action of microorganisms such as bacteria
and fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. In many cases, it will be preferable
to include isotonic agents, for example, sugars, polyalcohols such
as manitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought
about by including in the composition an agent which delays
absorption, for example, aluminum monostearate and gelatin.
[0112] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filter sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation include vacuum
drying and freeze-drying which yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0113] Formulations suitable for intra-articular administration can
be in the form of a sterile aqueous preparation of the drug that
can be in microcrystalline form, for example, in the form of an
aqueous microcrystalline suspension. Liposomal formulations or
biodegradable polymer systems can also be used to present the drug
for both intra-articular and ophthalmic administration.
[0114] Formulations suitable for topical administration, including
eye treatment, include liquid or semi-liquid preparations such as
liniments, lotions, gels, applicants, oil-in-water or water-in-oil
emulsions such as creams, ointments or pastes; or solutions or
suspensions such as drops. Formulations for topical administration
to the skin surface can be prepared by dispersing the drug with a
dermatologically acceptable carrier such as a lotion, cream,
ointment or soap. Particularly useful are carriers capable of
forming a film or layer over the skin to localize application and
inhibit removal. For topical administration to internal tissue
surfaces, the agent can be dispersed in a liquid tissue adhesive or
other substance known to enhance adsorption to a tissue surface.
For example, hydroxypropylcellulose or fibrinogen/thrombin
solutions can be used to advantage. Alternatively, tissue-coating
solutions, such as pectin-containing formulations can be used.
[0115] For inhalation treatments, inhalation of powder
(self-propelling or spray formulations) dispensed with a spray can,
a nebulizer, or an atomizer can be used. Such formulations can be
in the form of a fine powder for pulmonary administration from a
powder inhalation device or self-propelling powder-dispensing
formulations. In the case of self-propelling solution and spray
formulations, the effect can be achieved either by choice of a
valve having the desired spray characteristics (i.e., being capable
of producing a spray having the desired particle size) or by
incorporating the active ingredient as a suspended powder in
controlled particle size. For administration by inhalation, the
compounds also can be delivered in the form of an aerosol spray
from pressured container or dispenser which contains a suitable
propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
[0116] Systemic administration also can be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants generally are known in the art,
and include, for example, for transmucosal administration,
detergents and bile salts. Transmucosal administration can be
accomplished through the use of nasal sprays or suppositories. For
transdermal administration, the active compounds typically are
formulated into ointments, salves, gels, or creams as generally
known in the art.
[0117] The active compounds can be prepared with carriers that will
protect the compound against rapid elimination from the body, such
as a controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. Liposomal suspensions can
also be used as pharmaceutically acceptable carriers. These can be
prepared according to methods known to those skilled in the art,
for example, as described in U.S. Pat. No. 4,522,811.
[0118] Oral or parenteral compositions can be formulated in dosage
unit form for ease of administration and uniformity of dosage.
Dosage unit form refers to physically discrete units suited as
unitary dosages for the subject to be treated; each unit containing
a predetermined quantity of active compound calculated to produce
the desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals. Furthermore, administration can be by periodic
injections of a bolus, or can be made more continuous by
intravenous, intramuscular or intraperitoneal administration from
an external reservoir (e.g., an intravenous bag).
[0119] Where adhesion to a tissue surface is desired the
composition can include the drug dispersed in a fibrinogen-thrombin
composition or other bioadhesive. The inhibitor then can be
painted, sprayed or otherwise applied to the desired tissue
surface. Alternatively, the inhibitors can be formulated for
parenteral or oral administration to humans or other mammals, for
example, in therapeutically effective amounts, e.g., amounts that
provide appropriate concentrations of the inhibitor to target
tissue for a time sufficient to induce the desired effect.
[0120] The compounds of the present invention can be administered
directly to a tissue locus by applying the compound to a medical
device that is placed in contact with the tissue. An example of a
medical device is a stent, which contains or is coated with one or
more of the compounds of the present invention.
[0121] Active compound as identified or designed by the methods
described herein can be administered to individuals to treat
disorders prophylactically or therapeutically. In conjunction with
such treatment, pharmacogenomics (i.e., the study of the
relationship between an individual's genotype and that individual's
response to a foreign compound or drug) can be considered.
Differences in metabolism of therapeutics can lead to severe
toxicity or therapeutic failure by altering the relation between
dose and blood concentration of the pharmacologically active drug.
Thus, a physician or clinician can consider applying knowledge
obtained in relevant pharmacogenomics studies in determining
whether to administer a drug as well as tailoring the dosage and/or
therapeutic regimen of treatment with the drug.
[0122] In therapeutic use for treating, or combating, inflammation
and other immune-related diseases in mammals, the inhibitors or
pharmaceutical compositions thereof will be administered orally,
parenterally and/or topically at a dosage to obtain and maintain a
concentration, that is, an amount, or blood-level or tissue level
of active component in the animal undergoing treatment which will
be effective to modulate inflammation, e.g., by modulating the
migration of activated lymphocytes. Generally, an effective amount
of dosage of active component will be in the range of from about
0.1 to about 100, more preferably from about 1.0 to about 50 mg/kg
of body weight/day. The amount administered will also likely depend
on such variables as the type and extent of disease or indication
to be treated, the overall health status of the particular patient,
the relative biological efficacy of the compound delivered, the
formulation of the drug, the presence and types of excipients in
the formulation, and the route of administration. Also, it is to be
understood that the initial dosage administered can be increased
beyond the above upper level in order to rapidly achieve the
desired blood-level or tissue level, or the initial dosage can be
smaller than the optimum and the daily dosage can be progressively
increased during the course of treatment depending on the
particular situation. If desired, the daily dose can also be
divided into multiple doses for administration, for example, two to
four times per day.
[0123] All cited publications, patents, patent applications,
sequence information cited by GenBank, Ensembl or other public
sequence database accession numbers, and all other references that
are included in the attached papers and/or manuscripts are
specifically incorporated by reference herein in their entirety in
order to more fully describe the state of the known art pertaining
to the present invention.
[0124] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Materials and Methods
[0125] Materials. Recombinant mouse CXCL12 was obtained from
R&D Systems (Minneapolis, Minn.), forskolin and RpcAMPS from
Biomol (Plymouth Meeting, Pa.), and 3-isobutyl-1-methylxanthine
(IBMX), dipyridamole, Con A, dibutyryl cAMP and adenosine deaminase
type X from Sigma-Aldrich (St. Louis, Mo.). The PDE3 inhibitor
motapizone, the PDE4 inhibitor piclamilast and a PDE7-selective
inhibitor were supplied by colleagues.
[0126] Isolation of murine splenocytes. Splenocytes were isolated
from 6-8 week old C57BL/6 mice obtained from Jackson Laboratories
(Bar Harbor, Me.). Spleens were removed and a single-cell
suspension prepared using 40 .mu.m cell strainers (Fisher
Scientific). Cells were washed with RPMI 1640 medium supplemented
with 5% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin
and 100 mg/ml streptomycin (all from GIBCO). Red blood cells were
lysed using standard lysis buffer (0.15M NH.sub.4Cl, 10 mM
KHCO.sub.3, 0.1 mM EDTA PH 7.4). Cells were then washed and used in
chemotaxis assays either as unstimulated cells or after stimulation
with Con A (31 g/ml) as indicated.
[0127] Preparation of Test Reagents. Forskolin, IBMX, Motapizone,
Piclamilast, and dipyridamole were dissolved as 1000.times. stock
solutions in 100% DMSO and diluted into the chemotaxis assays to
give a final DMSO concentration of 0.1%. This concentration of DMSO
had no effect on migration of splenocytes in the presence or
absence of CXCL12. CXCL12 was prepared as a 100 .mu.g/ml stock
solution in PBS+0.1% BSA, Con A was prepared as a stock of 2.5
mg/ml in PBS, and dibutyryl cAMP and Rp-cAMPS were prepared as 50
mM stock solutions in water, and these reagents were diluted
directly into the chemotaxis assays.
[0128] Chemotaxis Assay. Chemotaxis assays were done in 24-well
transwell plates with a pore size of 5 .mu.m (Costar, Corning).
Splenocytes were placed in medium at a concentration of
3.times.10.sup.6 cells/ml. Where test agents were used, splenocytes
were pretreated with the agent or vehicle for 45 to 60 min,
following which 100 .mu.l of the splenocyte suspension was placed
into the upper chamber of transwell plates, and the lower chamber
was filled with 600 .mu.l of medium. When added to induce
migration, CXCL12 (250 ng/ml) was added to the lower chamber only.
Other test reagents were added as indicated to both the upper and
lower chambers. After 4 hours of incubation at 37.degree. C. in 5%
CO2, transwell inserts were gently removed and the number of cells
that migrated into the lower chamber were counted by withdrawing
500 .mu.l of lower chamber medium, mixing it with 10 ml of buffer
and counting the cells on a Coulter Counter (Beckman Coulter Z
series). Where results are expressed as % of CXCL12-stimulated
migration, this was calculated as follows: (cells migrated in
presence of CXCL12 and test reagent--cells migrated in medium
alone)/(cells migrated in presence of CXCL12-cells migrated in
medium alone).times.100. Experimental points for all chemotaxis
assays were performed in triplicate.
[0129] Quantitative real-time RT-PCR. Total RNA was isolated from
unstimulated splenocytes and from splenocytes stimulated by Con A
for different lengths of time as indicated, using RNeasy mini kits
(Qiagen) according to the manufacturer's instructions. cDNA was
synthesized using M-MLV reverse transcriptase (Promega). Primers
were designed using ABI Primer Express Software v3.0. and
synthesized by Invitrogen Life Technologies. Quantitative real-time
RT-PCR 7 was performed using an ABI 7500 fast system and data
analyzed using 7500 fast system SDS software v3.0. Sets of primers
with the following sequences were used: PDE4B2 primer sequences,
forward: ACCTGAGCAACCCCACCAA (SEQ ID NO:1), reverse:
CCCCTCTCCCGTTCTTTGTC (SEQ ID NO:2); PDE7A primer sequences,
forward: TCAGCAGCAATCTTGATGCAA (SEQ ID NO:3), reverse:
AGAGGCTGGGCACTTCACAT (SEQ ID NO:4); PDE8A primer sequences,
forward: CCTGCAGCATTCCCAAGTC (SEQ ID NO:5), reverse:
TGTATAAGGTTAGGCAGGTCAA (SEQ ID NO:6); ribosomal protein L19 (RPL19)
primer sequences, forward: CCAAGAAGATTGACCGCCAT (SEQ ID NO:7),
reverse: CAGCTTGTGGATGTGCTCCAT (SEQ ID NO:8). Amplicon sizes were
100 bp.
[0130] Statistics. Data are plotted as the means.+-.S.D. of
replicate determinations. Statistical significance of experimental
conditions relative to control were analyzed by Student's t-test
and significance indicated by asterisks in the figure, with the p
values given in the legend.
[0131] Proliferation assays. Lymphocytes and splenocytes are
obtained from naive mice by separation of draining lymph nodes or
spleen using cell strainers. Cells are washed twice in serum-free
AIM-V culture medium and resuspended in AIM-V culture media
supplemented with 10.sup.-5 M 2-ME. For proliferation assays,
splenocytes (3.times.10.sup.5 cells/well) and lymph node cells
(2.5.times.10.sup.5 cells/well) are plated in triplicate cultures
in 96-well microtiter culture plates. Anti-CD3 and anti-CD28 mAb
are immobilized according to standard procedures. Culture plates
are incubated for 48 h at 37.degree. C. Proliferation is assessed
by measuring [.sup.3H]-thymidine incorporation added at 0.2
.mu.Ci/well for the last 16 h. At the end of the incubation period,
cells are harvested and radioisotope incorporation measured as an
index of lymphocyte proliferation in a betaplate liquid
scintillation counter (FIGS. 16A, B).
[0132] In FIGS. 16A and 16B, T cells of regional draining lymph
nodes from wild-type (CD26.sup.+/+) and CD26.sup.-/- mice were
isolated on day 11 or 12 following immunization with MOG p35-55.
Cells were cultured with the indicated concentrations of MOG p35-55
(FIG. 16A) or PWM (FIG. 16B) for 72 h, and T cell proliferation in
each culture was measured using [.sup.3H]-thymidine incorporation
assay. The cell proliferation is shown as stimulation index (SI;
mean.+-.SD) of four independent experiments (*p<0.05,
CD26.sup.+/+ vs. CD26.sup.-/- mice). [.sup.3H]-thymidine
incorporation in lymph node cells stimulated with 50 .mu.g/ml MOG
p35-55 was determined as 10,332.+-.4,590 cpm (CD26.sup.+/+) and
45,180.+-.7,530 cpm (CD26.sup.-/-) and in PWM-stimulated lymph node
cells as 80,482.+-.30, 130 cpm (CD26.sup.+/+) and 122,133.+-.37,
608 cpm (CD26.sup.-/-).
[0133] Cytokine measurements. For determination of cytokine
secretion, lymph node cells or splenocytes are cultured in AIM-V
medium supplemented with 10.sup.-5 M 2-ME. Cells are stimulated
with anti-CD3 and anti-CD28. Cell culture supernatants are
harvested after 48 h and stored at -70.degree. C. until cytokine
determination. TNF-.alpha., IFN-.gamma., IL-2, IL-4 and IL-10
concentrations of cell culture supernatants are determined with
specific enzyme-linked immunosorbent assays according to standard
procedures (FIGS. 16C-H). In FIGS. 16C-16H, T cells were isolated
from regional draining lymph nodes of wild-type (CD26.sup.+/+) and
CD26.sup.-/- mice on day 11 or 12 following immunization with MOG
p35-55. MOG-primed lymphocytes were cultured in presence of
different concentration of MOG p35-55. Cell culture supernatants
were collected 48 h later. Levels of IFN-g (FIG. 16C), IL-2 (FIG.
16D), TNF-a (FIG. 16E), IL-4 (FIG. 16F), IL-10 (FIG. 16G), and
latent TGF-b1 (FIG. 16H) in the cultures were measured by specific
ELISA. Cytokine production is shown as mean.+-.SEM of four
independent experiments (*p<0.05). PDE inhibitors are added in
selected cultures in concentrations as indicated in Table 3.
[0134] Intracellular cytokine staining. As an alternative approach,
intracellular cytokine staining for IFN-.gamma. and IL-17 is
determined, a recently discovered `signature` cytokine for
autoimmune T cells (Langrish, et al., J Exp Med, vol. 201:233-240
(2005); Cua, et al., Nature, vol. 421:744-748 (2003); and
Hofstetter et al., J Neuroimmunol, vol. 170:105-114 (2005)). Cells
are washed twice in PBS, fixed with paraformaldehyde (2%), and then
permeabilized using saponin (0.5%) for intracellular staining. The
following mAbs are used: PE-coupled rat anti-mouse IL-17
(TC11-18H10) titrated to >0.25 .mu.g mAb/10.sup.6 cells;
FITC-coupled anti-IFN-.gamma. (XMG1.2) titrated to >0.5 .mu.g
mAb/10.sup.6 cells. The level of background staining is assessed
using rat IgG1 (PE-R3-34) and rat IgG1 (FITC-R3-34) at >0.25
.mu.g/10.sup.6 cells and >0.5 .mu.g/10.sup.6 cells,
respectively.
[0135] Isolation of T cell subpopulations. CD4.sup.+ and CD8.sup.+
T cell populations are isolated from lymph node cell cultures using
commercial kits based on magnetic bead assays according to standard
procedures.
[0136] Flow chamber assays. To test PDE control of adhesion
strength of activated T cells to VCAM-1, a flow chamber assay
system is used. (See e.g., Cinamon and Alon, Methods Mol Biol, vol.
239:233-242 (2004); Cinamon and Alon, J Immunol Methods, vol.
273:53-62 (2003); and Cinamon et al., Nat Immunol, vol. 2:515-522
(2001)) Recombinant mouse VCAM-1 is mixed in coating medium (PBS
buffered with 20 mM sodium bicarbonate pH, 8.5) with a fixed amount
of carrier (2 mg/ml HSA) and adsorbed on polystyrene plates for 2 h
at 37.degree. C., alone or with the indicated amounts of intact or
heat-inactivated chemokines. Plates are washed and blocked with HSA
(20 mg/ml). VCAM-1/chemokine-coated substrates are assembled as the
lower wall of the flow chamber (260-mm gap) and extensively washed
with binding medium. The flow chamber is mounted on the stage of an
inverted phase contrast microscope. All flow experiments are
conducted at 37.degree. C. T cells are perfused at 10.sup.6
cells/ml through the chamber at indicated flow rates generated with
an automated syringe pump. The entire period of cell perfusion is
recorded on videotape with a long integration CCD video camera and
a time-lapse recording program. All cellular interactions with the
adhesive substrates are determined by manually tracking the motions
of individual cells along 0.9-mm field paths for 1 min.
[0137] To test PDE control of vascular recruitment of activated T
cells, a model barrier of bEND.3 cells grown to confluence on
fibronectin coated polystyrene dishes is used. To induce the
critical adhesion molecule VCAM-1, the monolayers are activated
with TNF-.alpha. (2 .mu.g/ml) for 2 h. Following activation, the
dish is secured on the lower level of a parallel plate laminar flow
apparatus which creates a 260 .mu.M vertical gap). Once assembled,
CXCL12 (100 ng/ml) or CCL19 (100 ng/ml) is perfused over the
monolayer for 5 min to immobilize chemokine followed by perfusion
of buffer to remove unabsorbed chemokine from the chamber system.
Purified T cell population of a previously determined type is then
allowed to accumulate under a low shear stress of 0.75-1.5
dyn/cm.sup.2 for 40-120 s followed by an increased shear stress to
2-10 dyn/cm.sup.2 for 10-20 min. Shear stress is generated by an
automated syringe pump attached to the chamber outlet port. The
perfusion period is recorded by real-time videomicroscopy, during
accumulation, and time-lapse videomicroscopy, during the higher
shear stress period. Motion analysis is then performed manually on
the time-lapse video according to the following criteria: (I)
initial tethers (defined as T cells that make rolling or stationary
contact with the endothelial surface during the period of
observation), (II) firm stationary adhesion (defined as T cells
that make initial tethers and then adhere firmly, remaining fixed
for the duration of the assay), (III) locomotion of cells on the
apical endothelial monolayer (defined as T cells that form
non-stationary firm adhesion to the endothelium), and (IV)
transmigration through the monolayer (defined as T cells that made
initial tethers, firmly adhere to the endothelium, undergo
locomotion, and then migrate beneath the surface of the endothelium
through an intercellular junction).
[0138] Immunoblot. Analysis of PDE expression by Western Immunoblot
methodology is performed as described in a recent publication
(Tiwari, et al., Biochem. Pharmacol, vol. 69:473-483 (2005)).
Primary antibodies against each of the 21 known genes encoding PDEs
are available commercially, e.g., from FabGennix Inc. (Shreveport,
La.).
[0139] Peptides. Myelin oligodendrocyte glycoprotein peptide 35-55
(MOG p35-55), corresponding to mouse sequence
(MEVGWYRSPFSRVVHLYRNGK, SEQ ID NO:9), is synthesized on a peptide
synthesizer by standard 9-fluorenylmethoxycarbonyl chemistry, and
purified by high-performance liquid chromatography (HPLC).
[0140] Induction of active EAE and clinical evaluation. Active EAE
is induced in 8-12 weeks old mice by immunization with MOG p35-55
in complete Freund's adjuvant. 200 .mu.g of MOG p35-55 peptide and
800 .mu.g of killed mycobacterium tuberculosis are emulsified in
CFA and injected subcutaneously by means of four injections over
the flanks. In addition, 200 ng of pertussis toxin dissolved in 200
.mu.l PBS is injected i.p. at the day of immunization and again the
day after. Mice are monitored daily for clinical signs of EAE, and
scored according to the following criteria: 0, no signs of disease;
0.5, partial tail weakness; 1, limp tail; 1.5, limp tail and slight
slowing of righting; 2, partial hind limb weakness and/or marked
slowing of righting; 2.5, dragging of hind limb(s) without complete
paralysis; 3, complete paralysis of at least one hind limb; 4,
severe forelimb weakness; 5, moribund or dead. Daily clinical
scores are calculated and presented as the average (mean) and
standard error of the mean (SEM) of all individual disease scores
in each group. Statistical comparison of disease severity by
clinical score is accomplished by calculating the mean clinical
score for each mouse from day of disease onset to day 60 and
performing a non-parametric Wilcoxon analysis.
[0141] CD26.sup.-/- mice show significantly higher clinical and
histopathologic severity scores of EAE compared to wt mice. (A)
Disease course of EAE (mean score.+-.SEM) in wt (CD26.sup.+/+) and
CD26.sup.-/- mice is plotted from day 0 to day 38 (each group n=27
combined from 3 independent experiments; p=0.048, CD26.sup.+/+ vs.
CD26.sup.-/- mice). The insert shows incidence of EAE in the
experimental groups. (B) Histopathologic analysis revealed an
increased number of meningeal (M), parenchymal (P) and total (T)
inflammatory foci in CD26.sup.-/- mice compared to wild-type
(CD26.sup.+/+) mice. Data represent mean number of inflammatory
foci.+-.SEM. The differences in the number of foci between the two
groups (each n=4) was statistically significant with p=0.0003 (M),
p=0.0097 (P) and p=0.0023 (T).
[0142] Histopathology. These studies focus on scoring of
inflammatory infiltrates in EAE. Selected mice are perfused with 20
ml cold PBS on day 14 after immunization. Brains and spinal cords
are extracted, fixed in 4% (w/v) paraformaldehyde and embedded in
paraffin. Sections were stained with haematoxylin and eosin. Brain,
thoracic and lumbar spinal cord sections are evaluated and
meningeal, parenchymal and total numbers of inflammatory foci are
determined by an examiner blinded to the treatment status of the
animal.
[0143] Statistical analysis of EAE severity: Statistical comparison
of clinical EAE disease severity is accomplished by performing a
Wilcoxon analysis for non-parametric data sets using SigmaStat
software. Histopathological data are analyzed with using the
unpaired two-tailed Student's t-test using SigmaStat software.
Example 2
CXCL12 Induces Migration of Murine Splenocytes
[0144] The studies provided herein demonstrated that cAMP
modulation of T cell migration indicates different intracellular
regulation between stimulated and unstimulated cells. Directed
migration of T cells to specific tissues is believed to play an
important role in lesion formation associated with inflammatory
diseases. To investigate cAMP signaling and PDE control of T cell
migration, broad modulators of the synthetic and degradative
enzymes that regulate cAMP were used in chemotaxis assays. Previous
studies had shown that chemotaxis of human T cells to several
stimuli, including CXCL12, could be inhibited by agents known to
stimulate the cAMP signaling pathway. T cells used in these
previous chemotaxis studies were, however, quiescent, unstimulated
cells. Inasmuch as it is now well accepted that pro-inflammatory T
cell populations that participate in transendothelial migration and
enter sites of inflammation represent activated effector/memory T
cells with a Th1/Th17 phenotype (Langrish, et al., J Exp Med, vol.
201:233-240 (2005); Cua, et al., Nature, vol. 421:744-748 (2003);
and Hofstetter et al., J Neuroimmunol, vol. 170:105-114 (2005)),
unstimulated and ConA-stimulated splenocytes were compared for
modulation of their chemotactic response by agents that stimulate
the cAMP signaling pathway.
[0145] Using the transwell assay system, CXCL12 (250 ng/ml)
stimulated the migration of both unstimulated and Con A-stimulated
mouse splenocytes (FIG. 1). Mouse splenocytes (3.times.10.sup.5),
isolated as indicated in the Methods described above, were placed
in the upper chamber insert of 24 well transwell plates and the
number of cells migrating to the lower chamber were counted after 4
hr, as described in the Methods. Where included, CXCL12 (250 ng/ml)
was added to the lower chamber only. Results represent the
mean.+-.S.D. of four separate experiments with experimental points
assayed in triplicate. *p<0.001, **p<0.02.
[0146] In response to CXCL12, there was a 6.2-fold increase in the
number of unstimulated splenocytes and a 2.5-fold increase in the
number of Con A-stimulated splenocytes migrating to the side of the
chamber containing CXCL12. The number of cells migrating to the
chamber containing CXCL12 was about the same for unstimulated and
Con A-stimulated splenocytes. The difference in fold stimulation
between the two cell populations was primarily due to increased
migration of Con A-stimulated splenocytes, relative to unstimulated
splenocytes, in the absence of CXCL12.
Example 3
Effect of cAMP Analogue Adenylyl Cyclase Activator and PDE
Inhibitors on CXCL12 Induced Splenocyte Chemotaxis
[0147] The cell permeable cAMP analogue, dibutyryl cAMP (500 .mu.M)
significantly inhibited CXCL12-induced migration of both
unstimulated and Con A-stimulated splenocytes by 54% and 29%
respectively (FIG. 2). Splenocytes isolated from mice were assayed
for migration in response to CXCL12 (250 ng/ml), either directly
(unstimulated) or following 48 hr of incubation with 3 .mu.g/ml Con
A (stimulated), as described in Methods. To test for effects of
dibutyryl cAMP, cells were pretreated with dibutyryl cAMP (500
.mu.M) for 45-60 min prior to beginning the chemotaxis assay, and
the assays were conducted with dibutyryl cAMP (db-cAMP) present
(500 .mu.M) in both the upper and lower chambers of the transwell
plates. Data plotted are derived from a single experiment performed
in triplicate. *p<0.001; **p<0.02.
[0148] In contrast, the responses of these two cell populations,
the unstimulated and Con A-stimulated splenocytes, to adenylyl
cyclase activation or PDE inhibition were quite different.
Forskolin (25 .mu.M), a direct activator of adenylyl cyclase,
inhibited CXCL12-induced splenocyte migration by 31%, but had no
effect at all on CXCL12-induced migration of Con A-stimulated
splenocytes (FIG. 3). Chemotaxis assay conditions were the same as
those described above in connection with FIG. 2 except that the
test agents used were forskolin (Fsk) (25 .mu.M) and IBMX (300
.mu.M). Results represent the mean.+-.S.D. of four separate
experiments assayed in triplicate. *p<0.005; **p<0.05.
[0149] Similar to what was seen with activation of adenylyl
cyclase, the non-specific PDE inhibitor, IBMX (300 .mu.M) also
produced a differential effect on CXCL12-induced migration of
unstimulated and Con A stimulated splenocytes. Whereas IBMX
inhibited migration of unstimulated splenocytes by 57%, it only
inhibited Con A-stimulated splenocyte migration by 21% (FIG. 3).
When IBMX and forskolin were added together, however, greater
inhibition was seen. In the presence of both forskolin and IBMX,
migration of unstimulated splenocytes was inhibited by 74% and Con
A-stimulated splenocytes by 66%.
[0150] Response of Con A-stimulated splenocytes to PDE gene family
selective inhibitors was also examined. Chemotaxis assay conditions
were the same as those described above in connection with FIG. 2
except that only Con A-stimulated splenocytes were used and the
test agents used were the PDE3-selective inhibitor motapizone (10
.mu.M), the PDE4-selective inhibitor piclamilast (1 .mu.M) and a
PDE7-selective inhibitor (not shown). Data plotted are derived from
a single experiment performed in triplicate. There was no
statistically significant effect of any of these PDE inhibitors in
this assay.
[0151] Thus, whereas the cell permeable cAMP analogue, dibutyryl
cAMP, readily inhibited chemotaxis of both cell populations, the
adenylyl cyclase activator, forskolin, and the general
non-selective PDE inhibitor, IBMX, inhibited migration of
unstimulated splenocytes, but either had no effect at all
(forskolin) or only a limited effect (IBMX) on activated cells,
except when added together. Since direct addition of an analogue of
cAMP inhibits migration of both cell types, whereas broad
modulators of the synthetic and degradative enzymes that regulate
cAMP primarily inhibited migration only of unstimulated cells, it
was concluded that unstimulated and activated splenocytes must
differ in the way in which they regulate cAMP within the cell.
Example 5
Effect of PDE Inhibitors on Adhesion and Migration of Activated T
Cells
[0152] In the studies described herein, dipyridamole, a PDE
inhibitor capable of inhibiting PDE8, blocked adhesion and
migration of activated T cells, but selective PDE4 inhibitors did
not.
[0153] In inflammation, T cell homing to most target tissues
requires an initial tethering step that leads to rolling in
postcapillary venules followed by a chemokine dependent activation
step which triggers firm adhesion and T cell emigration (diapedesis
or transendothelial migration (TEM)). To determine PDE control of T
cell adhesion to brain derived endothelium and chemotaxis, PDE
inhibitors were tested in vitro using adhesion (FIG. 11) and
chemotaxis assays (FIGS. 4 and 5). It has become increasingly
apparent in recent years that in response to stimuli, cAMP
elevations in cells occur in a directed spatial and temporal
manner, resulting in the formation of microdomains of localized
cAMP concentrations within the cell, and it is the regulation of
these localized domains of cAMP in physiologically important
compartments that regulate specific functions of the cell (Rich, et
al., Proc Natl Acad Sci USA, vol. 98:13049-13054 (2001)). It is now
established that one way microdomains of localized cAMP
concentrations are achieved in the cell is through selective
expression and compartmentalization of different isoforms of PDEs
(Mongillo, et al. Circ Res, vol. 95:67-75 (2004); Rich, et al., J
Gen Physiol, vol. 118:63-78 (2001) and Brunton, L. L. 2003. PDE4:
arrested at the border. Sci STKE 2003:PE44.). Since migration of
unstimulated splenocytes was readily inhibited by the general
non-selective PDE inhibitor, IBMX, and activated splenocytes were
far less affected by IBMX, it was hypothesized that activated
splenocytes express an IBMX insensitive PDE activity in a
functionally relevant cell compartment linked to regulation of cell
migration and adhesion, which limits the accumulation of cAMP in
that compartment in response to activators of adenylyl cyclase,
IBMX or PDE selective inhibitors targeted to IBMX-sensitive PDE
gene families. The non-specific PDE inhibitor IBMX inhibits all
known PDE gene families capable of hydrolyzing cAMP with the
possible exception of PDE8, since expressed forms of full length
cDNAs for PDE8 have been reported to be resistant to IBMX
inhibition (Soderling et al., Proc Natl Acad Sci USA, vol.
95:8991-8996 (1998); Fisher et al., Biochem Biophys Res Commun,
vol. 246:570-577 (1998); and Gamanuma, et al., Cell Signal, vol.
15:565-574 (2003)). Among a wide variety of PDE inhibitors tested
against expressed forms of PDE8A, only dipyridamole was found to
inhibit this enzyme, with reported IC.sub.50s in the range of 4-9
.mu.M (Soderling et al., Proc Natl Acad Sci USA, vol. 95:8991-8996
(1998); Fisher et al., Biochem Biophys Res Commun, vol. 246:570-577
(1998); and Gamanuma, et al., Cell Signal, vol. 15:565-574 (2003)).
Therefore, in order to determine the effects of inhibition of PDE8
on splenocyte adhesion and migration, the effects of dipyridamole
were tested in these systems.
[0154] The hypothesis was first tested using a series of
experiments determining T cell adhesion to vascular endothelial
cells. Confluent bEND.3 brain derived endothelial cell monolayers
were pretreated with 100 ng/ml TNF-.alpha.. Con A activated
splenocytes (5.times.10.sup.6 cells/ml) were labeled with 5 .mu.M
Calcein AM for 30 min at 37.degree. C. and 7.times.10.sup.5
splenocytes were suspended in medium in the presence of
dipyridamole or vehicle control and added to each well. Splenocytes
were allowed to adhere for 30 min at 37.degree. C. without shaking
after which nonadherent cells were removed by washing the wells
4.times. with prewarmed D-PBS. For analysis, 7.times.10.sup.5
calcein AM labeled cells were added to an empty well as a
representation of maximal possible fluorescence. The fluorescence
of each well was read by a Victor 3v microplate reader with a
fluorescein filter set. Background fluorescence (medium only) was
subtracted from each well and percentage of adherent cells that is
resistant to detachment under the conditions of washing the wells
was calculated as fluorescence of well in percent of maximal
fluorescence (without washing).
[0155] As shown in FIGS. 11A and 11B, adhesion to endothelial cells
of Con A-stimulated splenocytes was not inhibited at all by the
PDE4-selective inhibitor, piclamilast (1 .mu.M). This concentration
of the PDE4 family specific PDE inhibitor piclamilast used
maximally inhibited the PDE family it targets without losing
selectivity for inhibition of that given PDE gene family.
Additionally, a PDE7-selective inhibitor had no significant effect
on adhesion of Con A-stimulated splenocytes as well (data not
shown). Among a wide variety of PDE inhibitors tested against
expressed forms of PDE8A, only dipyridamole was found to inhibit
this enzyme, with reported IC.sub.50s in the range of 4-9 .mu.M
(Soderling et al., Proc Natl Acad Sci USA, vol. 95:8991-8996
(1998); Fisher et al., Biochem Biophys Res Commun, vol. 246:570-577
(1998); and Gamanuma, et al., Cell Signal, vol. 15:565-574 (2003)).
Therefore, in order to determine the effects of inhibition of PDE8
on splenocyte adhesion, the effects of dipyridamole were tested in
this system. As shown in FIGS. 11A and 11B, dipyridamole (100
.mu.M) inhibited adhesion of Con A-stimulated splenocytes by
50-54%. The percent of cells resistant to detachment was determined
by fluorometer (**p<0.001). Then, tests were conducted to
determine whether the reduction in adhesion of activated
splenocytes by dipyridamole could be reversed by an additional
chemokine signal. Besides its chemotactic properties, CXCL12 is
known to promote T cell adhesion to vascular endothelium and TEM
under flow conditions. However, when used in the static assay
system described herein, addition of CXCL12 did not increase T cell
resistance to detachment, nor did it overcome the inhibition by
dipyridamole (FIG. 11B). The presentation of CXCL12 had no
influence on these results as both CXCL12 absorbed (CXCL12a) to
endothelium as well as soluble CXCL12 (CXCL12s) did not enhance
splenocyte attachment to the bEND.3 cell monolayer.
[0156] Next, in order to determine the effects of inhibition of
PDE8 on splenocyte chemotaxis, the effects of dipyridamole were
tested in this system. As shown in FIG. 4, migration of Con
A-stimulated splenocytes was not inhibited at all by the
PDE3-selective inhibitor, motapizone (10 .mu.M), nor by the
PDE4-selective inhibitor, piclamilast (1 .mu.M). These
concentrations of the family specific PDE inhibitors used also
maximally inhibit the PDE family they target without losing
selectivity for inhibition of that given PDE gene family (Lerner
and Epstein. Biochem J, vol. 393:21-41 (2006) and Tenor, et al.,
Clin Exp Allergy, vol. 25:616-624 (1995)). Additionally, a
PDE7-selective inhibitor had no significant effect on migration of
Con A-stimulated splenocytes as well (data not shown). Of note, in
addition to the relative insensitivity to IBMX, migration of
activated splenocytes was also found to be resistant to inhibition
by gene family specific inhibitors targeted to PDE3, PDE4, and
PDE7. The inhibition of stimulated splenocytes seen following
combined addition of forskolin and IBMX could result from excessive
increases in cAMP causing spillover from one cellular compartment
to another, and overwhelming the normal cAMP degradative system in
the compartment responsible for regulation of migration. The
non-specific PDE inhibitor IBMX inhibits all known PDE gene
families capable of hydrolyzing cAMP with the possible exception of
PDE8, since expressed forms of full length cDNAs for PDE8 have been
reported to be resistant to IBMX inhibition (Soderling et al., Proc
Natl Acad Sci USA, vol. 95:8991-8996 (1998); Fisher et al., Biochem
Biophys Res Commun, vol. 246:570-577 (1998); and Gamanuma, et al.,
Cell Signal, vol. 15:565-574 (2003)).
[0157] As only dipyridamole was found to inhibit PDE8A (Soderling
et al., Proc Natl Acad Sci USA, vol. 95:8991-8996 (1998); Fisher et
al., Biochem Biophys Res Commun, vol. 246:570-577 (1998); and
Gamanuma, et al., Cell Signal, vol. 15:565-574 (2003)), the effect
of dipyridamole on splenocyte migration was tested. As shown in
FIG. 5, dipyridamole (100 .mu.M) inhibited the CXCL12-induced
migration of both unstimulated and Con A-stimulated splenocytes by
55% and 54%, respectively. Addition of forskolin (25 .mu.M) along
with dipyridamole further potentiated the inhibition of migration
of the two cell populations to 76% and 68%, respectively (FIG. 5).
Dipyridamole inhibition of CXCL12-stimulated chemotaxis was
reversed by the cAMP-dependent protein kinase (PKA) inhibitor,
Rp-cAMPS. Dipyridamole and rolipram were shown to inhibit
lipopolysaccharide-stimulated release of TNF-.alpha. from
peripheral blood mononuclear cells (Eigler et al., Scand J Immunol
45:132-139 (1997)). Addition of adenosine deaminase to the cultures
potentiated the release of TNF-.alpha. and attenuated the
inhibition of TNF-.alpha. release by rolipram, suggesting that the
effects of these agents on TNF-.alpha. release in this system are
mediated, at least in part, through extracellular adenosine that
accumulates in the cultures (Eigler et al., Scand J Immunol
45:132-139 (1997)).
[0158] The effects of adenosine deaminase on CXCL12-stimulated
splenocyte migration and its inhibition by dipyridamole were
tested. Adenosine deaminase by itself had no effect on splenocyte
migration (not shown), nor did it affect the dipyridamole
inhibition of migration. Thus, in support of the hypothesis, it was
found that dipyridamole profoundly inhibited chemotactic migration
of both unstimulated and stimulated splenocytes and that its
actions appeared to be independent of its ability to inhibit
adenosine uptake. Moreover, when forskolin was added along with
dipyridamole, to stimulate adenylyl cyclase, inhibition of
migration was potentiated, leading to as much as 70-80% inhibition
of migration of both unstimulated and stimulated cells, further
suggesting that the effects of dipyridamole on chemotaxis are
mediated by its stimulation of the cAMP signaling pathway through
inhibition of PDE. The concept that dipyridamole is working through
a cAMP mediated effect is also borne out by the reversal of the
dipyridamole effect that is seen with the PKA antagonist,
Rp-cAMPS.
[0159] The non-specific PDE inhibitor IBMX inhibits all known PDE
gene families capable of hydrolyzing cAMP with the possible
exception of PDE8, since expressed forms of full length cDNAs for
PDE8 have been reported to be resistant to IBMX inhibition
(Soderling, et al., Proc Natl Acad Sci USA, vol. 95: 8991-8996
(1998); Fisher, et al., Biochem Biophys Res Commun, vol. 246:
570-577 (1998); and Gamanuma, et al., Cell Signal, vol. 15: 565-574
(2003)). Among a wide variety of PDE inhibitors tested against
expressed forms of PDE8A, only dipyridamole was found to inhibit
this enzyme, with reported IC50s in the range of 4-9 .mu.M
(Soderling, et al., Proc Natl Acad Sci USA, vol. 95: 8991-8996
(1998); Fisher, et al., Biochem Biophys Res Commun, vol. 246:
570-577 (1998); and Gamanuma, et al., Cell Signal, vol. 15: 565-574
(2003)). Therefore, in order to determine the effects of inhibition
of PDE8 on splenocyte migration, the effects of dipyridamole were
tested in this system. Chemotaxis assay conditions were the same as
those described above in connection with FIG. 2 except that the
test agents used were dipyridamole (100 .mu.M) and dipyridamole
(100 .mu.M)+forskolin (Fsk) (25 .mu.M). Results represent the
mean.+-.S.D. of four separate experiments assayed in triplicate.
*p<0.002; **p<0.05.
[0160] As shown in FIG. 5, dipyridamole (100 .mu.M) inhibited the
CXCL12 induced migration of both unstimulated and Con A-stimulated
splenocytes by 55% and 54% respectively. Addition of forskolin (25
.mu.M) along with dipyridamole further potentiated the inhibition
of migration of the two cell populations to 76% and 68%
respectively (FIG. 5). Dipyridamole inhibition of CXCL12-stimulated
chemotaxis was reversed by the cAMP-dependent protein kinase (PKA)
inhibitor, Rp-cAMPS (FIG. 6A). To determine the effect of Rp-cAMPS,
chemotaxis assay conditions were the same as those described above
in connection with FIG. 2 except that the test agents used were
RpcAMPS (1 mM) and dipyridamole (Dipy) (100 .mu.M). Results
represent the mean.+-.S.D. of two separate experiments assayed in
triplicate. *p<0.001.
[0161] Dipyridamole and rolipram were shown to inhibit
lipopolysaccharide-stimulated release of tumor necrosis factor
(TNF) from peripheral blood mononuclear cells (Eigler, et al.,
Scand J Immunol, vol. 45: 132-139 (1997)). Addition of adenosine
deaminase to the cultures potentiated the release of TNF and
attenuated the inhibition of TNF release by rolipram, suggesting
that the effects of these agents on TNF release in this system are
mediated, at least in part, through extracellular adenosine that
accumulates in the cultures (Eigler, et al., Scand J Immunol, vol.
45: 132-139 (1997)). The effects of adenosine deaminase on
CXCL12-stimulated splenocyte migration and its inhibition by
dipyridamole were tested. Adenosine deaminase by itself had no
effect on splenocyte migration (not shown), nor did it affect the
dipyridamole inhibition of migration (FIG. 6B). In the
determination of the effect of adenosine deaminase, results shown
represent stimulated splenocytes. Test reagents used were
dipyridamole (Dipy) (100 .mu.M) and adenosine deaminase (A.D.) (1
U/ml).
Example 5
Expression of Splenocyte mRNA for PDE4B2, PDE7A1, and PDE8A1
Following Con A Stimulation
[0162] Previous studies focused on PDE4 isoforms as intracellular
targets for therapies in chronic inflammatory diseases. However,
dose-limiting side effects in humans, of which nausea and vomiting
are the most common, have hampered the clinical success of PDE4
isoforms. Thus, the studies presented herein were carried out to
overcome these limitations by identifying other PDE isoforms that
are expressed in immune cells and inhibition of these isoforms
provides the same or better therapeutic activity as the PDE4
isoforms alone, but does not incur dose-limiting side effects. In
the initial step of a complete analysis of PDE expression patterns
and functions in inflammatory cells, the expression of splenocyte
mRNA for PDE4B2, PDE7A 1, and PDE8A1 following Con A stimulation of
mouse splenocytes was investigated.
[0163] The insensitivity of Con A-stimulated splenocytes to
adenylyl cyclase activators and PDE inhibitors other than
dipyridamole, relative to unstimulated cells, could be explained by
an upregulation of PDE8 following Con A stimulation, which would
prevent cAMP accumulation in activated cells. In order to examine
this possibility, quantitative real-time RT-PCR was performed to
look at mRNA levels of PDE4B2, PDE7A1, and PDE8A1 following Con A
stimulation. Quantitative real-time RT-PCR was performed on mouse
splenocytes at different times following Con A stimulation as
described in the Methods provided above. Relative expression of
mRNA was calculated as follows: (amplification number of target
gene/amplification number of RPL19 housekeeping gene at a given
time point)/(that for zero time). Results represent the
mean.+-.S.D. of three separate experiments assayed in triplicate.
*p<0.05, **p<0.01.
[0164] As shown in FIG. 7, mRNA for PDE8A1 is induced 2.7-fold
within 8 hr following Con A stimulation. In contrast, PDE7A1 mRNA
did not increase at all following Con A stimulation, and PDE4B2
mRNA increased by only 1.5-fold at 4 hr and then returned to
baseline by 8 hr. Thus, as in human T cells, PDE8A1 is induced upon
activation in mouse T cells and is a target to suppress the
function of activated cells.
Example 6
The Effect of Dipyridamole (DP) Treatment on EAE
[0165] The studies provided herein demonstrated that dipyridamole
suppressee clinical signs of inflammation in vivo. The therapeutic
potential of selective PDE4 inhibitors in Th1/Th17-mediated
inflammatory diseases (Langrish, et al., J Exp Med, vol.
201:233-240 (2005); and Cua, et al., Nature, vol. 421:744-748
(2003)) has been widely studied (Giembycz, Proc Am Thorac Soc, vol.
2:326-333; discussion 340-321 (2005); Giembycz, Curr Opin
Pharmacol, vol. 5:238-244 (2005); Bender and Beavo. Pharmacol Rev,
vol. 58:488-520 (2006); Bernareggi, et al., Br J Pharmacol, vol.
128:327-336 (1999); Giembycz, Br J Clin Pharmacol, vol. 62:138-152
(2006); Sommer et al., J Neuroimmunol, vol. 79:54-61. (1997); Jung
et al., J Neuroimmunol, vol. 68:1-11 (1996); Moore et al., J
Pharmacol Exp Ther, vol. 319:63-72 (2006); Martinez et al., Brain
Res, vol. 846:265-267 (1999); Bielekova, et al., J Immunol, vol.
164:1117-1124 (2000); Lagente et al., Mem Inst Oswaldo Cruz, vol.
100 Suppl 1:131-136 (2005); and Ouagued et al., Pulm Pharmacol
Ther, vol. 18:49-54 (2005)). While successful preventive treatment
is well documented, data on reversal of chronic inflammation are
less conclusive (Giembycz, Curr Opin Pharmacol, vol. 5:238-244
(2005); Smith and D. Spina, Curr Opin Investig Drugs, vol.
6:1136-1141 (2005); Dyke and Montana, Expert Opin Investig Drugs,
vol. 11:1-13 (2002); Huang, et al., Curr Opin Chem Biol, vol.
5:432-438 (2001); Essayan, Biochem Pharmacol, vol. 57:965-973
(1999); Kroegel and Foerster, Expert Opin Investig Drugs, vol.
16:109-124 (2007); Folcik et al., J Neuroimmunol, vol. 97:119-128
(1999); and Dinter et al., J Neuroimmunol, vol. 108:136-146
(2000)). To study the role of PDE8 in inflammation in vivo,
experimental autoimmune encephalomyelitis (EAE) was induced in
C57BL/6 mice, an animal model widely used for the study of the
pathogenesis and therapy of the human disease multiple sclerosis
(Brocke, et al., Nature, vol. 379:343 (1996); Brocke et al., Proc
Natl Acad Sci USA 96:6896 (1999); and Steinbrecher, et al., J
Immunol 166:2041 (2001)). EAE models can be performed using mutant
and conventional mice (Liblau et al., Int Immunol, vol. 9:799-803
(1997); Preller et al., J Immunol, vol. 178(7):4632-40 (2007); and
Ferber et al., J Immunol, vol. 156:5-7 (1996)).
[0166] From day 0 until day 23, mice were injected with 2 mg of the
PDE inhibitor DP per mouse per day or 0.1% DMSO in PBS as vehicle
control as indicated in FIG. 8. DP is the only inhibitor known to
block PDE 8 in addition to other PDEs. When EAE developed following
immunization with myelin oligodendrocyte glycoprotein peptide
35-55, mice showed paralytic signs of the disease and were scored
(EAE score) according to standard protocols (Brocke, et al.,
Nature, vol. 379:343 (1996); and Brocke et al., Proc Natl Acad Sci
USA 96:6896 (1999)). Average EAE scores+SD values of all mice in
each experimental group are shown.
[0167] Daily injections of DP profoundly suppressed the development
of clinical signs of EAE in immunized mice. These data suggest that
targeting PDE8 is a promising approach for the development of
treatments in inflammatory autoimmune or allergic diseases such as
multiple sclerosis, rheumatoid arthritis, asthma and inflammatory
bowel disease.
[0168] To assess whether dipyridamole prevented EAE progression
after the first signs of clinical disease onset, dipyridamole (1
mg/mouse/day i.p. for the first 8 days and 2.times.1 mg/mouse/day
i.p. from day 9 to day 22) or vehicle control (0.1% DMSO in PBS)
were administered once the mean clinical EAE scores reached grade
1. Dipyridamole given at a dose of 2.times.1 mg/mouse/day i.p., but
not a single dose of 1 mg/mouse/day i.p., significantly decreased
the clinical severity of EAE compared with vehicle control
treatment.
Example 7
Identification of PDE Targets
[0169] The studies described herein are used to identify novel PDE
targets in anti-CD3 and anti-CD28 stimulated T cells treated with
selective PDE inhibitors from wildtype and PDE mutant subjects such
as mice.
[0170] The studies described herein are carried out to test the
hypothesis that if activated T cells contribute to the pathogenesis
of smoking associated inflammation in vivo, then suppression of
these T cells should improve or cure these inflammatory conditions.
It is clear from a number of clinical trials with PDE4 inhibitors
that targeting of activated T cells in vivo is not a trivial task,
especially once they have been activated and have become
effector/memory T cells. PDE4 activity accounts for the majority of
the total PDE activity in T cells, and selective PDE4 inhibitors
showed great therapeutic efficacy in animal studies. Despite these
observations, when used in clinical trials for asthma and other
inflammatory illnesses, PDE4 inhibitors showed limited success, and
consequently, none have yet been approved for clinical use. A
possible explanation for the limited efficacy of selective PDE4
inhibitors in clinical trials is provided by early findings of PDE
enzymatic activity in resting T cells in which isoforms in addition
to PDE4 were shown to be expressed (FIG. 10.; Table 1) (Lerner and
Epstein. Biochem J, vol. 393:21-41 (2006) and Tenor, et al., Clin
Exp Allergy, vol. 25:616-624 (1995)).
TABLE-US-00001 TABLE 1 PDE isoforms as potential regulators of cAMP
signaling in immune cells PDE Isoform Specificity cAMP Km cGMP Km
Immune cell expression 1B1 cAMP and cGMP 7-24 .mu.M 3 .mu.M
Activated T cells Dendritic cells 1B2 cAMP and cGMP 7-24 .mu.M 3
.mu.M Activated T cells Macrophages 3B cAMP and cGMP 0.2-0.5 .mu.M
0.02-0.2 .mu.M Peripheral blood T cells Peripheral blood B cells
Macrophages 4A cAMP 1-4 .mu.M N/A Activated T cells 4B Primary B
cells 4D Macrophages Dendritic cells Neutrophils 7A1 cAMP 0.03-0.2
.mu.M N/A Activated T cells 7A3 B cells Macrophages Neutrophils 8A1
cAMP 0.04-0.15 .mu.M N/A Activated T cells
[0171] Additionally, more recent studies report the long term
induction in activated T cells of the specific PDE isoforms, PDE1B1
(Jiang, et al., Proc Natl Acad Sci USA, vol. 93:11236-11241
(1996)), PDE1B2 (Bender, et al., Proc Natl Acad Sci USA, vol.
102:497-502 (2005)), PDE4A4 (Jiang, et al., Cell Biochem Biophys,
vol. 28:135-160 (1998)), PDE4D1/D2 (Jiang, et al., Cell Biochem
Biophys, vol. 28:135-160 (1998)), PDE4D3 (Jiang, et al., Cell
Biochem Biophys, vol. 28:135-160 (1998)), PDE7A1 (Li et al.,
Science, vol. 283:848-851 (1999)), PDE7A3 (Glavas et al., Proc Natl
Acad Sci USA, vol. 98:6319-6324 (2001)), and PDE8A1 (Glavas et al.,
Proc Natl Acad Sci USA, vol. 98:6319-6324 (2001)). Further,
translocation of PDE4A4, PDE4B2, and PDE4D1/D2 to lipid rafts
following T cell activation was also reported recently (Abrahamsen
et al., J Immunol, vol. 173:4847-4858 (2004)). As described herein,
PDE8 was identified as an additional and novel target for
inhibition of chemotaxis of activated splenocytes (FIGS. 4, 5 and
7) (Dong, et al., Biochem Biophys Res Commun, vol. 345:713-719
(2006)). Thus, the fundamental concern with selective PDE4
inhibitors is that different constitutive or induced PDE isoforms
in T cells control cAMP levels and reduce the efficacy of PDE4
inhibitors in vivo or require treatment doses that lead to
significant side effects. A possible strategy to overcome these
limitations is to inhibit other PDE isoforms that are expressed in
immune cells in the hope that therapeutic activity can be retained
while reducing the side effects. In order to take this approach, a
complete analysis of PDE expression patterns and functions in
inflammatory cells is necessary. As a first approach to address
this question, the expression of splenocyte mRNA for PDE4B2,
PDE7A1, and PDE8A1 following Con A stimulation of mouse splenocytes
was investigated (FIG. 7). However, since these experiments were
performed with bulk splenocyte cultures activated by mitogen, it
remains unresolved whether the results were unique for this cell
population and stimulation condition, and whether or not the same
PDE isoform spectrum is expressed in purified inflammatory effector
T cells. Based on these preliminary studies, this question is
resolved in vitro by analyzing expression of PDE1B, PDE3B, PDE4A,
B, D, PDE7A and PDE8A, B in anti-CD3 and anti-CD28 stimulated T
cells.
[0172] This experiment is carried out to determine in vitro
expression of PDE1B, PDE3B, PDE4A,B,D, PDE7A and PDE8A, B isoforms.
Expression of PDE genes is assayed and compared in activated T
cells reactive to myelin antigen with unstimulated and mitogen
activated T cells, using specific primers in qRT-PCR and antibodies
in immunoblot analysis. Detection of specific PDE genes and
proteins is performed using the methods described above in Example
1. In order to assess the kinetics of PDE expression in T cells
undergoing activation, mouse CD4.sup.+ and CD8.sup.+ T cells in
lymph node cells from C56BL/6 mice are purified after in vitro
stimulation with anti-CD3 and anti-CD28 mAbs at various time points
(FIG. 13). From these experiments, PDE isoforms that can play a
regulatory role in activated inflammatory T cell populations are
identified.
[0173] In addition, studies are provided to determine compensatory
PDE isoform induction in anti-CD3 and anti-CD28 stimulated T cells
from PDE.sup.-/- mice and cells treated with selective PDE
inhibitors. To assess the expression of PDE isoforms in
autoreactive T cells in response to genetic or pharmacologic
modulation, compensatory changes in expression of PDEs under these
experimental conditions are evaluated. In order to accomplish this
goal, anti-CD3 and anti-CD4 stimulated T cells from PDE.sup.-/-
mice or T cells treated with selective PDE inhibitors are isolated
and assayed for expression levels of PDE1B, PDE3B, PDE4A,B,D, PDE7A
and PDE8A,B isoforms by qRT-PCR and Western immunoblotting. Mouse
mutants used in the proposed experiments are listed in Table 2.
(Yang, et al., J Immunol, vol. 171:6414-6420 (2003); Jin et al.,
Methods Mol Biol, vol. 307:191-210 (2005); Ariga et al., J Immunol,
vol. 173:7531-7538 (2004); Choi et al., J Clin Invest 116:3240-3251
(2006); Reed et al., J Neurosci, vol. 22:5188-5197 (2002); and
Vasta et al., Proc Natl Acad Sci USA, vol. 103:19925-19930 (2006)).
Selective inhibitors for these and further studies are compiled in
Table 3. An outline of the experimental approach is given above
(FIG. 13).
TABLE-US-00002 TABLE 2 PDE.sup.-/- mice and observed phenotype
Disrupted Gene Phenotype PDE1B .uparw. locomotor activity PDE3B
Insulin resistance PDE4A, B, D (4A) .dwnarw. airway disease (4B)
.dwnarw. TNF-.alpha. 90% (4D) emesis, reduced growth PDE7A None
apparent PDE8A, B None apparent
TABLE-US-00003 TABLE 3 Selective PDE inhibitors used for in vitro
studies Name Potency (IC.sub.50) Concentration in vitro Vinpocetine
PDE1 (5-25 .mu.M) 10 .mu.M Cilostamide PDE3 (0.005 .mu.M) 3 .mu.M
Rolipram PDE4 (1 .mu.M) 10 .mu.M BYK308438 PDE4B2 (204 nM) 1 .mu.M
PDE7A1 (17 nM) Proprietary (Altana) PDE7 (17 nM) 1 .mu.M
Proprietary (Pfizer) PDE8 (160 nM) 1 .mu.M Dipyridamole PDE8 (4-9
.mu.M) 100 .mu.M
Example 8
Determination of Regulatory Functions of PDE Isoforms
[0174] The studies described herein are carried out to determine
the regulatory functions of novel PDE isoforms in anti-CD3 and
anti-CD28 stimulated T cells from wildtype and PDE mutant mice and
T cells treated with selective PDE inhibitors.
[0175] These studies test the hypothesis that if PDE isoforms are
induced during T cell activation or as part of a compensatory
response to genetic or pharmacologic modulation, their role can be
determined in assays measuring T cell functions related to
inflammation as depicted in (FIG. 14). To address this goal, test
are run in specific PDE.sup.-/- mice (Table 2) anti-CD3 and
anti-CD28 mAb dependent T cell stimulation in proliferation and
cytokine production assays. Similarly, the effect of selective PDE
inhibitors is examined (Table 3). These experiments are carried out
to determine which PDEs control distinct T cell effector functions,
and to which extent. An additional critical T cell function
addressed in these studies is T cell recruitment at a model of the
microvascular endothelium. Directed migration of blood-borne cells
to distinct target tissues plays an important role in numerous
physiologic and pathologic conditions. Blocking leukocyte
extravasation has a profound therapeutic effect on inflammatory
diseases that involve recruitment of pathogenic T cells (Steinman,
Cell, vol. 85:299-302 (1996); Zamvil and Steinman, Annu Rev
Immunol, vol. 8:579-621 (1990); Martin, et al., Annu Rev Immunol,
vol. 10:153-187 (1992); Martin et al., Nat Immunol, vol. 2:785-788
(2001); Hafler, J Clin Invest, vol. 113:788-794 (2004); Fox and
Ransohoff, Trends Immunol, vol. 25:632-636 (2004); Ransohoff et
al., Nat Rev Immunol, vol. 3:569-581 (2003); Feldmann and Steinman,
Nature, vol. 435:612-619 (2005); Steinman, Nat Rev Drug Discov,
vol. 4:510-518 (2005); and Steinman and Zamvil, Trends Immunol,
vol. 26:565-571 (2005)). The efficacy of anti-migratory drugs
targeting a number of different molecules has been confirmed in
vitro and in vivo. Natalizumab, for example, which blocks
.alpha.4.beta.1 integrin and thereby prevents attachment of
leukocytes to their endothelial ligand, vascular cell adhesion
molecule-1 (VCAM-1), is now an approved drug for the treatment of
MS and Crohn's disease, thus highlighting adhesion blocking
antibodies as a promising avenue for the development of
therapeutics effective in inflammatory diseases (Steinman, Cell,
vol. 85:299-302 (1996); Zamvil and Steinman, Annu Rev Immunol, vol.
8:579-621 (1990); Martin, et al., Annu Rev Immunol, vol. 10:153-187
(1992); Martin et al., Nat Immunol, vol. 2:785-788 (2001); Hafler,
J Clin Invest, vol. 113:788-794 (2004); Fox and Ransohoff, Trends
Immunol, vol. 25:632-636 (2004); Ransohoff et al., Nat Rev Immunol,
vol. 3:569-581 (2003); Feldmann and Steinman, Nature, vol.
435:612-619 (2005); Steinman, Nat Rev Drug Discov, vol. 4:510-518
(2005); and Steinman and Zamvil, Trends Immunol, vol. 26:565-571
(2005)). T cell homing to most target tissues requires an initial
tethering step that leads to rolling in postcapillary venules
followed by an activation step which triggers firm adhesion and T
cell emigration (diapedesis or transendothelial migration (TEM)).
This process can be divided into four distinct steps, (i.e. 1.
rolling and arrest; 2. chemotactic stimulation; 3. adhesion
strengthening, firm arrest; and 4. TEM). Each of these four steps
involves distinct molecular pathways whose unique combination
selectively enables specific subpopulations of T cells to migrate
to particular organs. Efficient T cell recruitment at specific
sites from blood through endothelium and ultimately, into the
underlying parenchyma, requires strengthening of overall cellular
adhesiveness (avidity) through precise regulation of at least one
of three major integrins: .alpha.4.beta.1, .alpha.L.beta.2, or
.alpha.4.beta.7. Specifically, the increase in cellular avidity
under the shear stress environment of the bloodstream depends on
subsecond changes in intrinsic affinity and the number (valency) of
the integrin-integrin ligand bonds, as well as the cytoskeletal
anchorage of the integrin molecule. At the vasculature, the
required signals for integrin strengthening are provided by ligand
engagement and immobilized chemokines. Regulation of T cell
functions by cAMP, including adhesion, polarization and chemotaxis,
is well documented. Early work demonstrated that cAMP, acting
through PKA, inhibits chemoattractant-triggered integrin-dependent
leukocyte adhesion, thus establishing an important role for the
cAMP-PKA pathway in modulating T cell recruitment (Laudanna, et
al., J Biol Chem, vol. 272:24141-24144 (1997)).
[0176] Determination of the PDE isoform control of T cell
activation and function is accomplished using proliferation assay,
cytokine assays, intracytoplasmic cytokine staining by flow
cytometry and T cell recruitment assay in flow chamber system.
Function of defined PDEs in regulating T cell activity is
determined by testing anti-CD3 and anti-CD28 stimulated T cells in
proliferation and cytokine production assays (Preller et al., J
Immunol, vol. 178(7):4632-40 (2007)). The role of PDE1B, PDE3B,
PDE4A,B,D, PDE7A and PDE8A,B genes in regulating cAMP-PKA-dependent
vascular T cell recruitment is determined by real time
videomicroscopy measuring rolling and arrest, activation and
adhesion strengthening and transendothelial migration under
physiologic shear stress in vitro (FIG. 15) (Cinamon and Alon,
Methods Mol Biol, vol. 239:233-242 (2004); Cinamon and Alon, J
Immunol Methods, vol. 273:53-62 (2003); Cinamon et al., Nat
Immunol, vol. 2:515-522 (2001); and Shamri et al., Nat Immunol,
vol. 6:497-506 (2005)). Specifically, proliferation and cytokine
production of anti-CD3 and anti-CD28 stimulated T cells are
performed as described for antigen-specific T cells).
[0177] In order to determine the role of PDEs in regulating T cell
recruitment at brain derived endothelial cells, the studies of
adhesion and chemotaxis are extended by analyzing T cell attachment
to VCAM-1 and endothelial cells and TEM in a flow chamber model.
During extravasation, T cells must adhere to and migrate across the
endothelial barrier under the shear stress environment created by
moving blood (Cinamon and Alon, Methods Mol Biol, vol. 239:233-242
(2004); Cinamon and Alon, J Immunol Methods, vol. 273:53-62 (2003);
Cinamon et al., Nat Immunol, vol. 2:515-522 (2001); and Shamri et
al., Nat Immunol, vol. 6:497-506 (2005)). Most migration studies
have been performed under static condition in modified Boyden
chambers such as Transwell assays. While T cells will migrate
across a barrier in response to a chemokine gradient, it is unknown
if the gradient mimics the in vivo chemokine distribution along the
endothelial surface. Furthermore, chamber assays are performed
under non-physiological timeframes. Intravital microscopy has
predicted the window of T cell extravasation to be on the order of
min, yet static migration assays are analyzed in h. Continuous
shear stress in vitro promotes rapid and efficient transmigration
of lymphocytes which is dependent on integrins, an intact actin
cytoskeleton, and chemokine signaling, thus providing a
physiologically relevant framework in which to interpret T cell
recruitment. The .alpha.4.beta.1 integrin/VCAM-1 interaction is
involved in each of the steps leading to recruitment of T cells to
the CNS, while globally increasing cAMP results in decreased T cell
adhesion and motility. It remains to be determined how tight
regulation of cAMP may differentially affect the distinct
recruitment steps. Several PDEs are known to be compartmentalized
intracellularly and thus degrade cAMP in a spatially restricted
manner. As a result of this, even PDEs less globally abundant than
PDE4 may well be functionally critical. While the role of PDE4 in T
cell migration has been addressed, none of these experiments has
been performed under physiologic shear stress conditions.
Therefore, w the role of all PDEs expressed in T cells in addition
to PDE4 is determined using the flow chamber assay system.
Example 9
Identifying Unique and Overlapping Functions of PDE1, PDE3, PDE4,
PDE7 and PDE8 Gene Families During Inflammation
[0178] Studies are provided herein to determine the effect of
non-PDE4 selective PDE inhibitors in the treatment of established
inflammation. For example, knock-out of specific PDE genes and
injection of selective PDE inhibitors could control T cell
proliferation or cytokine production and migration, but fail to
suppress inflammatory T cells in vivo. Therefore, studies are
carried out to determine the susceptibility of specific PDE.sup.-/-
mice to inflammation in vivo. For this purpose, active EAE is
induced, and this model is used to test therapeutic efficacy of
selective PDE inhibitors to abolish or ameliorate established
clinical signs of this inflammatory disease (Giembycz, Curr Opin
Pharmacol, vol. 5:238-244 (2005); Smith and D. Spina, Curr Opin
Investig Drugs, vol. 6:1136-1141 (2005); Dyke and Montana, Expert
Opin Investig Drugs, vol. 11:1-13 (2002); Huang, et al., Curr Opin
Chem Biol, vol. 5:432-438 (2001); Essayan, Biochem Pharmacol, vol.
57:965-973 (1999); Kroegel and Foerster, Expert Opin Investig
Drugs, vol. 16:109-124 (2007); Folcik et al., J Neuroimmunol, vol.
97:119-128 (1999); and Dinter et al., J Neuroimmunol, vol.
108:136-146 (2000)). EAE is used because it represents a
well-characterized model system to test anti-inflammatory therapies
in vivo (Steinman, Cell, vol. 85:299-302 (1996); Zamvil and
Steinman, Annu Rev Immunol, vol. 8:579-621 (1990); Martin, et al.,
Annu Rev Immunol, vol. 10:153-187 (1992); Martin et al., Nat
Immunol, vol. 2:785-788 (2001); Hafler, J Clin Invest, vol.
113:788-794 (2004); Fox and Ransohoff, Trends Immunol, vol.
25:632-636 (2004); Ransohoff et al., Nat Rev Immunol, vol.
3:569-581 (2003); Feldmann and Steinman, Nature, vol. 435:612-619
(2005); Steinman, Nat Rev Drug Discov, vol. 4:510-518 (2005); and
Steinman and Zamvil, Trends Immunol, vol. 26:565-571 (2005); and
Steinman and Zamvil, Ann Neurol, vol. 60:12-21 (2006)).
[0179] Specific PDE.sup.-/- mice and wt control mice are immunized
with the MOG p35-55 in CFA and PTX to induce EAE (Preller et al., J
Immunol, vol. 178(7):4632-40 (2007)). Animals are observed for up
to 60 days for clinical signs of EAE. Selected animals at days 5-50
and all mice at day 60 are sacrificed and the brain and spinal cord
examined by H&E histology to determine whether PDE genes
control EAE. As a complementary approach, C57BL/6 wt mice are
immunized with MOG p35-55 to induce EAE, and the selective PDE
inhibitors are injected after the onset of clinical signs of EAE
(FIG. 16; usually day 10-15). Dosage of inhibitors used are listed
in Table 4.
TABLE-US-00004 TABLE 4 Selective PDE inhibitors used in in vivo
studies Name Dose/day Vehicle Route Specificity Vinpocetine 3 mg/kg
PBS and i.p. PDE1 Tween20 Cilostamide 10 mg/kg PBS i.p. PDE3
Rolipram 1-6.25 mg/kg PEG, saline i.p. PDE4 Proprietary 1 mg/kg
DMSO i.p. PDE7 (Altana) Proprietary 1 mg/kg DMSO i.p. PDE8 (Pfizer)
Dipyridamole 100 mg/kg DMSO i.p. PDE8
[0180] In initial experiments, animals are treated for a period of
10, 20 and 30 days to determine whether non-PDE4 selective PDE
inhibitors treat established EAE, and whether this is a long-term
treatment effect or requires continuous application of PDE
inhibitor. EAE is induced, and then studies are carried out to test
whether PDE4 and non-PDE4 selective inhibitors treat EAE in an
additive or synergistic fashion. This is tested by injection of
selective PDE4 inhibitors with each one of the selective PDE1,
PDE3, PDE7 and PDE8 inhibitors for the treatment of EAE.
[0181] Objective outcome measure is clinical EAE score and
histopathology of CNS tissue. C57BL/6 wt mice are susceptible to
active EAE. It has been established treatment of C57BL/6 mice with
dipyridamole, a PDE8 inhibitor (FIG. 7). If PDE mutant mice, as
expected for PDE4.sup.-/- and PDE8.sup.-/- mice, display altered
clinical course of EAE as compared to wt control mice, their
peripheral immune system is examined by quantifying CD4+ and
CD8.sup.+ T cell populations in the spleen and lymph nodes. This
alternative approach is used for any mutant strain with an EAE
phenotype. The studies described above are carried out to indicate
the composition of peripheral T cell populations as attempts are
made to purify T cells for the study of PDE expression and
functions in vitro. Based on published reports, there is little
evidence for PDE gene knock out affecting overall T cell
development and compartment in mice (Yang, et al., J Immunol, vol.
171:6414-6420 (2003); Jin et al., Methods Mol Biol, vol.
307:191-210 (2005); Ariga et al., J Immunol, vol. 173:7531-7538
(2004); Choi et al., J Clin Invest 116:3240-3251 (2006); Reed et
al., J Neurosci, vol. 22:5188-5197 (2002); and Vasta et al., Proc
Natl Acad Sci USA, vol. 103:19925-19930 (2006)). If it is
determined that T cells from PDE.sup.-/- mice or inhibitor treated
T cells fail to display any obvious T cell dysfunctions in vitro in
the studies described above, it is still possible for PDE gene
knock out or PDE inhibitors to prevent or treat EAE, for example by
immune deviation or induction of suppressive or regulatory T cells
(Tregs) in vivo. This possibility is supported by a recent report
indicating signals required to induce and maintain Tregs include
Foxp3-dependent repression of PDE3B (Gavin, et al., Nature,
ePublication, (Jan. 14, 2007); vol. 445(7129):771-5 (2007)). If the
studies described above produce diverging results from in vitro and
in vivo studies, additional studies are carried out to address
mechanisms of tolerance induction (Steinman, Cell, vol. 85:299-302
(1996); Zamvil and Steinman, Annu Rev Immunol, vol. 8:579-621
(1990); Martin, et al., Annu Rev Immunol, vol. 10:153-187 (1992);
Martin et al., Nat Immunol, vol. 2:785-788 (2001); Hafler, J Clin
Invest, vol. 113:788-794 (2004); Fox and Ransohoff, Trends Immunol,
vol. 25:632-636 (2004); Ransohoff et al., Nat Rev Immunol, vol.
3:569-581 (2003); Feldmann and Steinman, Nature, vol. 435:612-619
(2005); Steinman, Nat Rev Drug Discov, vol. 4:510-518 (2005); and
Steinman and Zamvil, Trends Immunol, vol. 26:565-571 (2005)) by PDE
knock out or selective inhibition through effector T cell
suppression, deviation Rocken et al., Immunol Today, vol.
17:225-231 (1996); Rocken and Shevach, Immunol Rev, vol.
149:175-194 (1996); and Racke et al., J Exp Med, vol. 180:1961-1966
(1994), deletion or energy, or generation of Tregs (Qiao, et al.,
Immunology, vol. 120(4):447-55 (2007); and Shevach et al., Immunol
Rev 212:60-73 (2006)).
Example 10
Effect of Targeting PDE8 on Dosage Requirements for PDE4
Inhibition
[0182] The efficacy of rolipram in reversing established EAE and
preventing EAE progression is unclear (Moore et al., J Pharmacol
Exp Ther 319:63-72 (2006)). Several selective PDE4 inhibitors,
including cilomilast and roflumilast, are in clinical trials for
the treatment of chronic obstructive pulmonary disease. Despite
some encouraging data from these phase III clinical trials, the
current generation of PDE4 inhibitors is hampered by a low
therapeutic index. A major obstacle is their propensity to evoke
side effects, of which nausea, diarrhea, abdominal pain, vomiting,
increased gastric secretions, and dyspepsia are the most common
(Burnouf and Pruniaux. Curr Pharm Des 8:1255-1296 (2002)).
Therefore, the therapeutic potential of inhibiting PDE isoforms
additional to PDE4 are investigated using the studies carried out
herein. One means of improving the therapeutic index and safety of
PDE4 inhibitors may lie in targeting PDE isoforms in addition to
PDE4 (Giembycz, Curr Opin Pharmacol 5:238-244 (2005); Giembycz,
Proc Am Thorac Soc 2:326-333; discussion 340-321 (2005); Giembycz,
Br J Clin Pharmacol 62:138-152 (2006); and Giembycz and Smith, Curr
Pharm Des 12:3207-3220 (2006). Inhibition of PDE4 with PDE7 and/or
PDE8 enhances clinical efficacy. Using inhibitors to additional
PDEs that are effective in treating EAE, a combinational treatment
is developed to limit the dose of PDE4 inhibitor needed, thus
maintaining efficacy while limiting side effects. In order to
achieve this goal, studies are carried out to address the role of
PDE isoforms in CNS inflammation in vivo, using EAE as a suitable
in vivo disease model. In these studies, therapeutic doses of
selective PDE4 inhibitors along with PDE7 and PDE8 inhibitors are
titrated in the EAE model. In the EAE model, various dosages of a
PDE inhibitor, alone or in combination with other PDE inhibitors,
are administered as shown in FIG. 17.
Example 11
Inhibition of Metastatic Cancers Using Selective PDE Inhibitors
[0183] The studies below are carried out in relation to breast
cancer metastases; however, the methods and compositions used
herein are also useful in the treatment and/or inhibition of a wide
variety of metastatic cancers.
[0184] Deaths from breast cancer almost always arise from
metastasis of the transformed cells to other sites in the body
(Steeg Nat Med, 12: 895-904, 2006). Hence, uncovering a means of
inhibiting breast cancer metastasis would provide a significant
advance in the treatment of this disease. Studies in cell lines and
animals have shown that breast cancer cell growth and migration can
be inhibited by cAMP (Marko et al., Chem Res Toxicol, 13: 944-948,
2000; O'Connor et al., J Cell Biol, 143: 1749-1760, 1998; and
O'Connor et al., J Cell Biol, 148: 253-258, 2000). Selective
elevation of cAMP in breast cancer cells is, therefore, an
effective means to treat this disease, either alone, or in
combination with other established treatments. A principal means of
selectively elevating cAMP and activating the cAMP signaling
pathway in a given tissue type is through inhibition of selective
form(s) of cAMP phosphodiesterase (PDE) present in that tissue.
With the existence of multiple transcription initiation sites, as
well as alternatively spliced forms of many of these PDE genes,
more than 100 different forms of PDE have been identified, many of
which have been shown to vary in their expression in different
tissues, intracellular localization, and interaction with different
intracellular signaling pathways (Lerner et al., Biochem J, 393:
21-41, 2006; Bender and Beavo, Pharmacol Rev, 58: 488-520, 2006;
Soderling and Beavo, Curr Opin Cell Biol, 12: 174-179, 2000; and
Francis et al., Prog Nucleic Acid Res Mol Biol, 65: 1-52, 2001).
Where PDE expression has been examined in human breast cancer cell
lines, such as MCF-7, by activity analysis, results show PDE4
activity to be the predominant form expressed Marko et al., Chem
Res Toxicol, 13: 944-948, 2000). Treatment of some breast cancer
cell lines with PDE4 selective inhibitors inhibits both their
growth and their migration and induces them to undergo apoptosis
(Marko et al., Chem Res Toxicol, 13: 944-948, 2000; O'Connor et
al., J Cell Biol, 143: 1749-1760, 1998; and O'Connor et al., J Cell
Biol, 148: 253-258, 2000). The PDE4 gene family consists of four
homologous, but distinct genes, encoding at least 20 different
splice variants (Houslay, Drug Discov Today, 10: 1503-1519, 2005).
Several recent studies have shown that the chemokine, CXCL12,
acting through its cognate receptor, CXCR4, regulates the
directional trafficking and invasion of breast cancer cells to
sites of metastasis (Luker and Lucker, Cancer Lett, 238: 30-41,
2006; Smith et al., Cancer Res, 64: 8604-8612, 2004; Fernandis et
al., Oncogene, 23: 157-167, 2004; and Lee et al., Mol Cancer Res,
2: 327-338, 2004). A recent study has shown that the newly
discovered PDE gene, PDE8A, regulates the CXCL12-induced chemotaxis
of activated lymphocytes, and that it is necessary to inhibit PDE8,
as well as PDE4, in order to inhibit CXCL12-directed chemotaxis of
activated lymphocytes (Dong, et al., Biochem Biophys Res Commun,
345: 713-719, 2006). Hence, PDEs provide excellent targets for
inhibiting breast cancer metastasis. The studies herein analyze PDE
expression in cultured human breast cancer cells, and through
selective inhibition of these PDE forms, using gene family-specific
PDE inhibitors and RNAi techniques, to identify targets for
inhibiting migration of the breast cancer cells.
[0185] The studies herein test the hypothesis that PDE inhibitors,
acting through PKA-dependent phosphorylation and inhibition of
RhoA, inhibit breast cancer cell motility, and thereby inhibit
breast cancer metastasis. Breast cancer cell motility is dependent
upon activation of the small GTPase protein, RhoA, and stress fiber
formation. RhoA is inhibited upon phosphorylation by cAMP-dependent
protein kinase (PKA), following stimulation of the cAMP signaling
pathway. Stimulation of the cAMP signaling pathway is achieved by
inhibiting the form(s) of PDE expressed in breast cancer cells. The
effects of this stimulation on breast cancer motility is
analyzed.
[0186] These studies analyze the expression of different forms of
PDEs in breast cancer cells, using both estrogen receptor positive
and negative cell lines, and 2) examine the effect of selective
inhibition or suppression of the expression of specific PDE forms
expressed in these breast cancer cell lines for their effect on a)
migration of the cells, b) formation of GTP-activated RhoA, and c)
stress fiber formation. Analysis of cAMP PDE isoforms expressed in
breast cancer cell lines is determined both by quantitative
real-time RT-PCR and Western immunoblotting, using antibodies
specific to each of the known cAMP PDE genes. Inhibition of the
expressed forms of PDE with selective PDE gene-family inhibitors,
or suppression of their expression with RNAi, is examined for their
effects on migration of the cells, GTP-activated RhoA formation,
and stress fiber formation.
[0187] To analyze the expression of different forms of PDEs in
breast cancer, two breast cancer cell lines, MCF-7, an estrogen
receptor-positive cell line, and MDA-MB-231, an estrogen
receptor-negative cell line, are used. Quantitative analysis of the
expression of PDEs in these cell lines is determined by
quantitative real-time RT-PCR (qPCR) and Western immunoblot
analysis using probes and antibodies specific for the known genes
of cAMP PDEs. Of the 21 known genes that encode PDEs, 5 are
specific for cGMP, and 16 are capable of hydrolyzing cAMP. Analysis
is concentrated on the expression of these 16 cAMP PDE genes,
notably PDEs 1A, B, C, 2A, 3A, B, 4A, B, C, D, 7A, B, 8A, B, 10A,
11A. qPCR and Western immunoblot determination of PDE expression
are measured by methods recently published for determination of PDE
expression in hematopoietic cells (Dong, et al., Biochem Biophys
Res Commun, 345: 713-719, 2006; and Tiwari et al., Biochem.
Pharmacol., 69: 473-483, 2005). Antibodies for all 21 PDE genes
have now been developed and are available commercially from
FabGennix Inc., Shreveport, La., and where positive expression of
mRNA for cAMP PDEs is seen, an examination of corresponding protein
expression is as well, using antibodies purchased from this
company.
[0188] To test the effects of inhibition of the expressed PDEs on
migration of the breast cancer cells in culture, PDE inhibitors are
tested for their effects on breast cancer cell migration in
response to CXCL12 using a transwell assay system (Dong, et al.,
Biochem Biophys Res Commun, 345: 713-719, 2006). Dose responses and
time courses are performed with the inhibitors. Initial studies
focus on inhibition of PDE4, since PDE4 has been reported to be
expressed in breast cancer cells, based on activity analyses and
also because PDE4 inhibitors have already been shown to be targets
for inhibition of breast cancer metastasis. Four very selective and
very potent PDE4 inhibitors are used rolipram, RO 20-1724,
piclamilast (RP73401), and roflumilast. It has been observed, for
example, that piclamilast inhibits purified PDE4 with a
Ki.apprxeq.0.3 nM, and does not begin to inhibit any of the other
PDE gene families until concentrations of more than 5 orders of
magnitude higher are reached. Although these inhibitors are very
selective for PDE4 relative to the other PDE gene families, they do
not distinguish between the four PDE4 subtypes, PDEs 4A-D, which is
most likely due to the high degree of sequence homology in the
catalytic region of these PDE4 genes. Several years ago the
catalytic region of PDE4B was crystallized and its structure
determined by X-ray diffraction analysis (Xu et al., Science, 288:
1822-1825, 2000). Subsequent to this, an additional crystallization
and structural analysis was accomplished with PDE4 bound to the
inhibitor, rolipram (Xu et al., J Mol Biol, 337: 355-365, 2004).
Using this published knowledge, a scaffold-based drug discovery
platform based on the 3-dimensional structure of the catalytic site
of PDE4B was developed to enable screening for novel inhibitors
that showed specificity for inhibition PDE4B over that of the other
PDE4 subtypes. This approach produced several PDE4B selective
inhibitors, including PLX513, which shows 10-20-fold greater
inhibitory specificity for PDE4B over PDE4A, C and D (Card et al.,
Nat Biotechnol, 23: 201-207, 2005). Indeed, in a recent study of
highly malignant human lymphomas that overexpress PDE4B, PLX513 was
dramatically more effective than rolipram in inducing apoptosis of
these cells (Smith et al., Blood, 105: 308-316, 2005). Therefore,
PLX513 and any other available PDE4 subtype selective inhibitors
are tested in the system described herein. In addition to these
PDE4 inhibitors, selective inhibitors for all other PDE gene
families that expressed in breast cancer cells are also tested for
the effect on cell migration as well. The effect of PDE on cell
migration is evaluated by knocking down its expression with RNAi
directed against it (Lynch et al., J Biol Chem, 280: 33178-33189,
2005). For those PDE gene families for which pharmacological
inhibition has a positive effect on inhibiting migration, RNAi
methodology is used to determine which subtypes and which expressed
splice variants of a given PDE gene family are the important
targets for this inhibition, as was done recently to define which
splice variants of PDE4 are functionally important in regulating
.beta.-arrestin-mediated .beta.-adrenergic receptor desensitization
(Lynch et al., J Biol Chem, 280: 33178-33189, 2005).
[0189] Migration of the breast cancer cells in response to CXCL12
and lysophosphatidic acid, as compared to control cells without a
stimulus, is assayed by the transwell method (Dong, et al., Biochem
Biophys Res Commun, 345: 713-719, 2006), with modifications for
these adherent cells as described (Bender and Beavo, Pharmacol Rev,
58: 488-520, 2006; Soderling and Beavo, Curr Opin Cell Biol, 12:
174-179, 2000). Dose responses and time courses for inhibition of
this migration by inhibitors of the different PDEs expressed in the
cells, added alone and in combination, is assessed. Chemotaxis
assays are done in 24-well transwell plates with a pore size of 8
.mu.m, with the lower surface of the membrane in each transwell
chamber coated for 30 min with 15 .mu.g/ml laminin-1. Cells are
harvested with trypsin and rinsed in serum-free media. Lower
transwell chambers contain either medium alone (control), CXCL12
(250 ng/ml), or lysophosphatidic acid (100 nM). Cells
(5.times.10.sup.4) suspended in DME/BSA media are added to the
upper chamber. Following 4 h incubation at 37.degree. C.,
non-migrating cells are removed from the upper chamber with a
cotton swab and cells that had migrated to the lower surface of the
membrane are fixed with 100% methanol and stained with 0.2% crystal
violet in 2% ethanol and counted.
[0190] PKA has been reported to phosphorylate and inactivate RhoA
(Howe, Biochim Biophys Acta, 1692: 159-174, 2004; Qiao et al., Am J
Physiol Lung Cell Mol Physiol, 284. L972-980, 2003 and Chen et al.,
Exp Biol Med (Maywood), 230: 731-741, 2005), and treatment of
fibroblasts with the PDE4 inhibitor, rolipram, leads to a sharp
decrease in the activated state of GTP-bound RhoA in the cells
(Fleming et al., J Cell Sci, 117: 2377-2388, 2004). Tests are
carried out to determine if PDE inhibitors cause a similar decrease
in the activated state of RhoA in breast cancer cells. To determine
this, breast cancer cells are plated onto laminin in the presence
and absence of PDE inhibitors for 1 hr before being lysed. Lysates
from these cells are incubated with a bacterially produced fusion
protein, GST-C21, to bind GTP-RhoA, bound to glutathione-coupled
agarose beads. The beads are washed in lysis buffer, eluted in
SDS-PAGE sample buffer and the amount of bound RhoA determined by
Western blotting. These experiments are carried out to determine if
inhibition of specific expressed PDEs leads to prevention of the
activation of RhoA in the cells.
[0191] The major driving force of migration is the extension of a
leading edge protrusion or lamellipodium, the establishment of new
adhesion sites at the front, cell body contraction, and detachment
of adhesions at the rear. All these steps involve the assembly, the
disassembly or the reorganization of the actin cytoskeleton, and
each must be coordinated both in space and time to generate
productive, net forward movement (Hall, Science, 279: 509-514,
1998; and Raftopoulou and Hall, Dev Biol, 265: 23-32, 2004).
Therefore, if inhibition of PDE and its subsequent inhibition of
RhoA are important for the inhibition of the migration of breast
cancer cells, this should be reflected in an effect on the
formation of stress fibers and focal adhesion structures. As such,
the effect of PDE inhibitors on stress fibers and focal adhesion
structures, in cells grown in the presence and absence of laminin
and stimulated with CXCL12 or vehicle alone (control), is evaluated
by visualizing these with fluorescent staining, respectively, of
actin and vinculin. For visualizing stress fibers and focal
adhesion structures, cells are fixed with 5% paraformaldehyde,
washed with PBS supplemented with 100 mM glycine, then
permeabilized with PBS supplemented with 0.1% saponin and 20 mM
glycine. After blocking with PBS supplemented with 0.1% saponin and
10% FCS for 1 hour at room temperature, cells are incubated with
primary antibody to vinculin at a dilution of 1:100 The vinculin is
then visualized with a species-specific fluorescein isothiocyanate
(FITC)-coupled secondary antibody (dilution 1:100; Sigma). Actin
filaments are visualized with tetramethylrhodamine
.beta.-isothiocyanate (TRITC)-phalloidin (1 mg/ml for 45 minutes)
(Sigma). Cells are then visualized using the microscopes in the
imaging core. These experiments are carried out to determine if PDE
inhibitors have effects on stress fiber and focal adhesion
structure formation to account for their inhibition of breast
cancer migration.
[0192] All publications and patent documents cited herein are
incorporated herein by reference as if each such publication or
document was specifically and individually indicated to be
incorporated herein by reference.
[0193] While the invention has been described with reference to
preferred embodiments, it will be understood by those skilled in
the art that various changes may be made and equivalents may be
substituted for the elements thereof without departing from the
scope of the invention. In addition, many modifications may be made
to adapt the teaching of the invention to a particular situation,
population, individual or diagnostic or treatment method without
departing from the essential scope thereof. Therefore, it is
intended that the invention not be limited to the particular
embodiments and best mode contemplated for carrying out this
invention as described herein.
* * * * *